WO2003083088A2 - Stem cell therapy - Google Patents

Abstract

A method of providing a primary germ layer cell for use in implant/transplant therapy, comprising providing a cell mass comprising an endoderm cell, an ectoderm cell and/or a mesoderm cell, and separating from the cell mass a primary germ layer cell selected from endoderm, ectoderm and mesoderm cells.

Description

STEM CELL THERAPY

FIELD OF THE INVENTION

This invention relates to a method to produce precursors of differentiated cells and their therapeutic use in tissue engineering and cell/tissue transplantation.

BACKGROUND OF THE INVENTION

Animal embryonic development combines cell proliferation and cell/tissue differentiation to produce an intact organism and is a highly regulated developmental process. Mammalian embryonic development is remarkably conserved during the early stages. Post fertilisation the early embryo completes four rounds of cleavage to form a morula of 16 cells. After several more rounds of division these cells then develop into a blastocyst. Cells in a blastocyst can be divided into two distinct regions: the inner cell mass, which will form the embryo, and the trophectoderm, which will form extra-embryonic tissue such as the placenta.

Cells that form part of the embryo up until the formation of the blastocyst are totipotent. This means that each cell has the developmental potential to form a complete embryo and all the cells required to support the growth and development of that embryo. In contradistinction, a pluripotent or multipotent cell has the developmental potential to form a variety of tissues. During blastocyst formation, the cells that comprise the inner cell mass are said to be pluripotential.

A blastocyst undergoes further development to give rise to a blastula which comprises the three primary germ layers (ectoderm, endoderm and mesoderm). It is from these germ layers that the principal cell types of the body are derived, e.g., nervous, epithelial, connective and muscular cells. It is known which germ layer each body cell type is derived from:

These principal cell types (endoderm, mesoderm and ectoderm) represent the primary trends of differentiation from the earliest generalised cells of the embryo and their appearance, which differs more and more as they multiply to form the first embryonic tissues, is marked by a selective reduction in the potentialities coded in the DNA content of their nuclei. The different levels of cells are however not absolutely fixed and under certain conditions cells may alter their characteristics surprisingly.

Pluripotent embryonic cells can be isolated from two embryonic sources. Cells derived from the inner cell mass of the blastocyst formed during early embryogenesis are termed embryonic stem cells (ES cells). Embryonic germ cells (EG cells) are pluripotent cells collected from fetal tissue at a somewhat later stage of development, i.e., primordial germ cells isolated from the mesenteries or genital ridges of days 8.5-12.5 post coitum embryos which would ultimately differentiate into germ cells. As used herein the term pluripotential cell refers equally to ES and/or EG cells. A useful review of stem cells is Odorico J S et al, Stem Ce//s2001; 19: 193-204, which is included herein by reference.

Each of these types of pluripotent cell has the same developmental potential with respect to differentiation into alternate cell types. Importantly, pluripotential cells have an increased commitment to terminal differentiation when compared to a totipotent cell. An intact embryo cannot be produced from a single ES or EG pluripotential cell.

Adult stem cells obtained from mature tissues differentiate into a narrower range of cell types. Accordingly many cells of medical interest might not be obtained from adult-born stem cells. It is also less feasible to develop large-scale cultures from adult stem cells.

A potentially limitless source of cells, stem cells can both duplicate themselves and produce differentiated cell types that constitute the various tissues or organ systems of the human body. The use of in vitro cultures of pluripotential stem cells and their tissue-committed derivatives, particularly human cells, has important ramifications in transplantation and/or replacement therapies (regenerative medicine) for critical tissues which have been damaged through injury or disease. Because of their pluripotent nature, ES and EG cells can differentiate into a variety of cell types and/or tissues and organs. Potential applications include, by no way of limitation, treatment of various severe pathological conditions such as neurodegenerative diseases (neuronal reconstitution in Parkinson's, Alzheimer, MS and ALS), neurological and neurosensorial pathological conditions (spinal cord injury, cerebral ischemia/stroke, retina diseases), endocrine metabolic diseases (islet body reconstitution in diabetes, pituitary diseases, hepatic failure) and cardiovascular diseases (myocardial tissue reconstitution post-MI, cardiac heart failure and coronary heart diseases).

It is therefore proposed that ES and EG cells will be directed to differentiate to specific cell types. The standardized production of large, purified populations of normal human cells such as heart muscle cells and neurons will provide a potentially limitless source of cells for drug discovery and transplantation therapies. Cells that have already been observed to differentiate from human ES cell lines have included gut epithelium (endoderm), cartilage, bone, and smooth and striated muscle (mesoderm); and neural epithelium, embryonic ganglia, and stratified squamous epithelium (ectoderm). Thomson et al, Science 1998 Nov 6;282(5391): 1145-1147. It is considered that, in order to realise the full potential of human pluripotent stem cells, the conditions for directed, lineage-restricted differentiation of ES cells must be defined. Studies to date on ES cell differentiation in vitro rely primarily on the selection and enrichment of specific lineages from the many that may be present when cell differentiation is induced. Also, strategies must be developed to obtain the large numbers of pure populations of differentiated cells that would"- be required ^• for engraftments (John Gearhart, Science 1998 Nov 6;282(5391):1061 - 1062). Odorico J S et al, Stem Cells 2001;19:193-204, teach that an early step in the establishment of successful stem cell therapy will be to purify a particular cell type of interest from a mixed population.

Munsie M J et al (Curr Biol 2000 Aug 24;10(16):989-92 and WO 00/67568) have reported the isolation of pluripotent murine stem cells from reprogrammed adult somatic cell nuclei. Embryos were generated by direct injection of mechanically isolated cumulus cell nuclei into mature oocytes by piezo-assisted micromanipulation. The donor nucleus was placed into an intact recipient cell to produce an aneuploid cell, which was maintained for a period to allow the donor nucleus to be reprogrammed before the recipient cell nucleus or nuclear DNA was removed or destroyed. In other embodiments of the Munsie technique, the recipient cell is enucleated substantially simultaneously with introduction of the donor nucleus, eg the latter is introduced substantially immediately before enucleation. In any event, the reprogrammed cell may be used to generate cells, cell lines, tissues, organs, embryos and animals. WO 00/67568 is included herein by reference, in particular but not exclusively example 2 thereof.

Munsie et al describe that embryonic stem (ES) cells isolated from cumulus-cell-derived blastocysts displayed the characteristic mo hology and marker expression of conventional ES cells and underwent extensive differentiation into cell types derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm) in tumours and in chimaeric foetuses and pups. The ES cells were also shown to differentiate readily into neurons and muscle in culture.

This study shows that pluripotent stem cells can be derived from nuclei of terminally differentiated adult somatic cells and offers a model system for the development of therapies that rely on autologous, human pluripotent stem cells. ES cells derived from somatic cell transplantation could restore function to diseased or damaged tissues, or be genetically altered before transplantation to deliver gene therapy. Transplantation studies in the mouse have shown that ES-cell derived cardiomocytes, neural precursors, haematopoietic precursors and insulin-secreting cells can survive and function in recipient animals. Nevertheless, according to Munsie et al, achievement of this goal requires the establishment of robust pluripotent stem cell culture and in vitro differentiation systems in order to establish a pluripotent cell-line which, upon exposure to various differentiation factors, can lead to the production of selected differentiated tissue for use, twter alia, in transplantation therapy, in vitro cultures of human ES/EG cells must be established. This has proved to be particularly problematical.

US 5 453 357 and US 5 690 926, both included ^"herein by reference, describe the establishment of in vitro cultures of ES/EG cells which are derived from non-murine species. Typically the ES/EG cultures have well defined characteristics. These include, but are not limited to; (i) maintenance in culture for at least 20 passages when maintained on fibroblast feeder layers; (ii) production of clusters of cells or 'embryoid bodies' in culture; (iii) ability to differentiate into multiple cell types in monolayer culture; (iv) formation of embryo chimeras when mixed with an embryo host; (v) expression of ES/EG cell specific markers.

WO 96/22362, which is included herein by reference, discloses cell-lines and growth conditions which allow the continuous proliferation of primate ES cells and provides the first indication that conditions which allow the establishment of human ES/EG cells in culture may be determined. The ES^' cells disclosed in WO 96/22362 exhibit a range of characteristics or markers associated with their pluripotential characteristics such as the expression of specific cell markers SSEA-1 (1), SSEA-3 (+), SSEA-4 (+), TRA-1-60 (+), and TRA-1-81 (+) (Shevinsky et al 1982; Kannagi et al 1983; Andrews et al 1984), alkaline phosphatase (+) and combinations thereof. The established primate cell-lines have stable karyotypes and continue to proliferate in an undifferentiated state ^"in continuous culture. Importantly the primate ES cell-lines also retain the ability, throughout their continuous culture, to form tissues derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm).

Pluripotential cells are also characterised by a characteristic chromosomal methylation pattern. The eukaryotic genome is variably methylated through the addition of methyl (-CH₃) groups which are attached to cytosine residues in DNA to form 5'methylcytosine ( 5'-mC). Methylation is correlated with the control of gene expression whereby genes that are hypomethylated tend to be highly expressed. Hypermethylation is correlated with reduced gene expression. Pluripotential cells will have a typical methylation pattern which may be analysed at a genomic level or at the level of a specific gene. Thomson et al (Science 1998 Nov 6;282(5391):1145-1147) disclose conditions in which human ES cells can be established in culture. The human cell-lines share the above characteristics which are shown by primate ES cells and show high levels of telomerase activity which confers the ability of continuous division in culture. Telomerase enzymes add, de novo, repetitive DNA sequences to the ends of chromosomes. These ends are referred to as telomeres. The telomeres of human chromosomes^" contain the sequence ⁷5 TTAGGG 3' repeated approximately 1000 times at their ends. In young, dividing cells the telomeres are relatively long. In ageing, or non dividing cells, the telomeres become shortened and there is a strong correlation between telomere shortening and proliferative capacity. Methods to increase telomere length and proliferative capacity are described in WO9513383. Telomerase enzymes and telomerase activity are constitutively highly expressed in haematopoietic stem cells of cord origin.

The establishment of human EG cell cultures is disclosed in WO 98/43679. EG cells were isolated from the gonadal or genital ridges of human embryos and were found to exhibit continuous proliferation in culture in an undifferentiated state, normal karyotype and the ability to differentiate into selected tissues under defined conditions.

It is well known that amphibian somatic cell nuclei retain their ability to give rise to entire embryos when transplanted into enucleated oocytes (Gurdon 1974). Determination of the pluripotency of these cells must be controlled by the egg cytoplasm which can 'reprogram' the somatic cell nucleus into a totipotent state. This effect has also been observed in the transfer of mammalian somatic cell nuclei to enucleated oocytes wherein the nuclei retain this plasticity and can be reprogrammed, (Campbell et al., Wakayama et al). The material produced is genetically identical to the somatic cell donor.

GB 2318578 and corresponding specifications US 6147276 and WO 97/07669 disclose the use of nuclei from differentiated or partially differentiated cells in nuclear transfer wherein gene expression of the transferred nuclei is reprogrammed to 'bring out' their inherent totipotency. GB 2318578 discloses the use of quiescent cells, i.e., those which are not actively proliferating by means of the mitotic cell cycle, as nuclear donors in the reconstitution of an animal embryo. Changes that occur in the donor nucleus which are observed after embryo reconstruction and which are required for efficient nuclear transfer are induced in the nuclei of cells prior to their use as nuclear donors by causing them to enter the quiescent state. Quiescent cells have a very limited proliferative potential due to their inherent chronological status (absence of telomerase activity).

In contrast to the present invention, GB 2318578 and its corresponding specifications are concerned with embryo reconstitution and production of whole organisms therefrom. The document highlights the difficulties in promoting development to term following the use of ES cells in nuclear transfer. These- patent documents describe the technique used to produce the famous cloned sheep called "Dolly" and this technique is conveniently referred to as "Dolly technology".

US 6147276 is included herein by reference, in particular but not exclusively examples 1 through 5 thereof.

Other studies have emphasised the role of cell cycle co-ordination of the donor nucleus and recipient cytoplasm in the development of embryos reconstructed by nuclear transfer (Campbell et al., Biol. Reprod. 49933-942 (1993) and Biol. Reprod. 50 1385-1393 (1994) ).

The mitotic cell cycle comprises four distinct phases; GI, S, G2 and M. Initiation of the cell cycle (start) occurs in the GI phase and it is here that the decision to undergo another cell cycle is made. The remainder of the GI phase is the pre-DNA synthesis phase. DNA synthesis takes place in the S phase and is followed by the G2 phase, which is the period between DNA synthesis and mitosis respectively. Mitosis occurs at M phase followed by cytokinesis. Quiescent cells, however, are those which are not actively dividing and are described as being in the GO state.

A number of metabolic changes have been reported in quiescent cells. These include monophosphorylated histones, ciliated centrioles, reduction or complete cessation in all protein synthesis, increased proteolysis, decrease in transcription and increased turnover of RNA resulting in a reduction in total cell RNA, disaggregation of polyribosomes. accumulation of inactive 80S ribosomes and chromatin condensation (reviewed Whitfield et al, Control of Animal Cell Proliferation, 1 331-365 (1985).

In our International patent application No PCT/GB01/04229 we describe a method for forming a pluripotent cell, the method comprising transferring a hematopoietic stem cell nucleus into a recipient oocyte and, before or after transfer, removing the oocyte nucleus. We describe that such a pluripotent cell may be used for the manufacture of a cell mass comprising ectodermal, mesodermal or endodermal cells for use in implant/transplant therapy: development of such a cell provides for the formation of a cell mass from which a blastula-type mass comprising ectodermal, mesodermal and/or endodermal cells may be derived.

Ectodermal, mesodermal and/or endodermal cells derived from such a blastula-type mass are used for implant/transplant therapy by their direct introduction into the patient to be treated. Late-stage differentiation of the cell or cells into the required tissue type occurs in-vivo by orthotopic implant (e.g. ectodermal-derived stem cells into CNS/PNS).

In one embodiment of the invention, the ectodermal, mesodermal, endodermal cell or cell-line may be autologous to the patient requiring treatment. Most preferably, the hematopoietic stem cell or the stem cell nucleus is obtained at birth of the subject and cryopreserved prior to use in the method.

The present invention provides the direct use of ectodermal, mesodermal and/or endodermal cell(s) in implant/transplant therapy. The implanted/transplanted cell(s) are implanted into tissue derived from primary germ cells of the same type (ectoderm, mesoderm or endoderm) as the implanted cells and differentiation of the cell or cells into the required tissue type occurs in- vivo.

The term "primary germ lεtyer cell" includes not only cells derived from a primary germ layer of ablastula but also cells from other sources and of the same type as blastula germ layer cells, ie having substantially the characteristics of natural primary germ layer cells.

The implanted cells (normally but not invariably more than one cell will be used) may be derived from any suitable source. Typically, one or more pluripotent or totipotent cells are placed in conditions suitable to form a cell mass having primary germ layer cells. Such a cell mass, which may conveniently be referred to as a blastoid cell mass, may be derived from in vitro fertilisation or formed from stem cells, for example ES cells, EG cells or cells obtained by nuclear transplantation. The stem cells are differentiated into precursor cells, either in vitro or in an in vivo or ex vivo environment, for example following injection into a host blastocyst or implantation into a host animal. In one class of embodiments the primary germ layer cells are derived by transferring a hematopoietic stem cell nucleus into a recipient oocyte and, before or after transfer, removing the oocyte nucleus to form a pluripotent cell and using the pluripotent cell for the manufacture of a cell mass comprising ectodermal, mesodermal or endodermal cells (ie cells of those types). In another class of embodiments the cells are not primary germ layer cells derived from nuclear transfer into an oocyte of an HSC nucleus.

The method of the present invention allows for post-natal orthotopic (e.g. neural-committed stem cells injected into diseased CNS/PNS areas, or endocrino-committed stem cells injected into diseased endocrine organs) autologous (self) reconstitution or allogeneic cell/tissue transplant (donor to recipient) to achieve in vivo anatomo-functional reconstitution. In one embodiment of the invention, the primary germ layer cells are used for post-natal autologous (self) reconstitution; the nucleus which is used for the nuclear transfer process is derived from cells, e.g. cryopreserved cells, of the same individual requiring the therapeutic intervention; in some methods these cells are a cryopreserved set of HSC from the individual and in other methods they are not. Suitably, the nucleus is extracted from a somatic cell of the individual, for example from a skin biopsy, and then injected into an enucleated oocyte.

Where the implanted/transplanted cells are an exact genetic match to the patient receiving the cells, no immune rejection is present and thus no immune suppression is required for a long lasting engraftment (the oocyte antigenic counterpart is diluted throughout divisions and will represent a minor antigenic pattern within the more differentiated cells).

It is well known that allogeneic material, if transplanted into another individual, may illicit a severe immune reaction in the host and thus be destroyed unless adequate, though toxic, immunosuppressive regimens are employed. Those methods of the present invention in which the implanted transplanted cells are derived from an HSC donor nucleus provide the important advantage that, due to developmental immaturity and higher plasticity of the HSC, the material for use in transplant is less immunogenic and with greater proliferative nature. This provides for a greater rate of success with allogeneic cell tissue transplant for which a certain critical number of committed stem cells might be warranted to obtain a successful tissue reconstitution. The allogeneic route may be preferred in circumstances where an individual has a genetic predisposition to disease and autologous (self) material cannot be used. Some examples of the therapeutic applications and value of this approach are the establishment, through the above technology, of autologous dopaminergic neurons or pre-neurons to be used for neurosurgical transplantation into the degenerated substantia nigra of patients with Parkinson's disease; the establishment of autologous neurons in other neurodegenerative disorders (Multiple Sclerosis, ALS); the establishment of autologous neurons in patients with spinal cord injury; the replacement of CNS neurons lost following stroke. Other areas of critical intervention are the establishment of autologous Langerhans beta-cells producing insulin or entire islet of Langerhans producing counteracting hormones such as glucagon in patients with diabetes and the establishment of striated myocardial cells by the injection, through coronary catheterisation, of mesoderm cells into an acute area of myocardial infarction.

STATEMENTS OF THE INVENTION

In one aspect, the invention provides the use of a cell or a cell mass for the preparation of a primary germ layer cell (endoderm-type cell, ectoderm-type cell or mesoderm-type cell) for use in implant/transplant therapy.

The cells may be of human or non-human origin. The cells may be for therapy of a human or of a non-human animal.

The initial cell or cells may be obtained by transfer of a nucleus to a recipient cell. In one class of methods the nuclear transfer does not comprise transfer of a haematopoietic stem cell nucleus to an oocyte. In other methods, the nuclear transfer does comprise transfer of a haematopoietic stem cell nucleus to an oocyte, as taught in PCT/GB01/04229.

Suitably, the nuclear transfer comprises transfer of the donor nucleus to an enucleated oocyte. The nuclear transfer may alternatively comprise transfer of a donor nucleus, eg haematopoietic stem cell nucleus, to a recipient cell to produce an aneuploid cell, maintaining the aneuploid cell in a suitable environment for a period sufficient to allow the donor nucleus to be reprogrammed, and generating a reprogrammed diploid cell from said reprogrammed aneuploid cell by removal, destruction or loss of the recipient cell nucleus or nuclear DNA from said reprogrammed aneuploid cell. The invention also includes uses wherein the initial cell or cells is/are obtained by in vitro fertilisation.

Another aspect of the invention resides in a method of providing a primary germ layer cell for use in implant/transplant therapy, comprising providing a cell mass comprising an endoderm cell, an ectoderm cell and/or a mesoderm cell, and separating from the cell mass a primary germ layer cell selected "from endoderm, ectoderm and mesoderm cells. Usually, a plurality of cells of the same type are separated from the cell mass.

In some methods of the invention, the cell mass is prepared by nuclear transfer of a nucleus into an enucleated oocyte and providing conditions which are suitable for the division of the oocyte to a cell mass containing primary germ layer cells, eg a blastula-like stage.

In other methods of the invention, the cell mass is prepared by "nuclear addition" as described in WO 00/67658 and providing conditions which are suitable for the division of the oocyte to a cell mass containing primary germ layer cells, eg a blastula-like stage.

In those methods involving nuclear transfer, the donor cell may be quiescent or non-quiescent (e.g. a haematopoietic stem cell from peripheral blood, bone marrow or from cryopreserved cord blood stem cells^'). In one class of methods, the donor cell is not a haematopoietic stem cell. In another class of methods, the donor cell is a haematopoietic stem cell.

The invention further provides a primary germ layer cell or a cell-line derived from a primary germ layer cell provided by a method of the invention.

In a further aspect there is provided a method to treat conditions or diseases potentially capable of benefiting from tissue and/or organ transplantation comprising introducing into a patient to be treated a primary germ layer cell of the invention and optionally a suitable excipient, diluent or carrier, the primary germ layer cell being introduced into tissue of the corresponding type to the introduced cells.

Another method of the invention comprises a method of implant/transplant therapy comprising performing a method of providing a primary germ layer cell as described herein and introducing into a patient to be treated a primary germ layer cell derived from said method and optionally a suitable excipient, diluent or carrier, the cell being introduced into tissue of the corresponding type to the introduced cell.

In some methods, a haematopoietic stem cell is obtained at birth of the patient and cryopreserved prior to use of the cell as a nuclear donor in the preparation of the introduced precursor cell.

In other methods a haematopoietic stem cell nucleus is obtained from the patient and then cryopreserved prior to use of the nucleus in the method of the invention.

The introduced precursor cell is allogeneic to the patient or, preferably, autologous to the patient.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the invention will now be described by example only and with reference to the following materials and methods.

The essence of the invention resides in the use of a primary germ layer cell in tissue engineering, also known as implant or transplant therapy. Thus, a key aspect of the invention resides in the use of a cell product (a cell or cell mass) for the preparation of a primary germ layer cell for use in implant/transplant therapy.

The manner in which the primary germ layer cell is obtained is not critical to the invention but it is usually provided using nuclear transfer technology. The invention will therefore be described, for illustrative purposes, with reference to a non-limiting procedure which commences with nuclear transfer. Such a procedure comprises the following steps: (i) nuclear transfer; (ii) cell division to form a cell mass containing one or more primary germ layer cells; (iii) separation of primary germ layer cell; and (iv) implant (also called "transplant") of primary germ layer cell into patient.

1. Nuclear transfer Nuclear transfer may be carried out by any technique which reprogramm.es cells to form a pluripotent or totipotent cell. Suitable techniques include the Dolly technology and the technique described by Munsie et al, which is sometimes referred to as "nuclear addition".

The Dolly technology comprises transferring the nucleus of a quiescent diploid donor cell into a recipient cell (in practice an enucleated oocyte,- preferably an enucleated metaphase π oocyte, inactivated oocyte or preactivated oocyte). Nuclear transfer is suitably effected by fusion through electrical stimulation ("electrofusion"). Before or after transfer, or concomitantly with it, the recipient cell is stimulated into development by parthenogenic activation, for example by the application of one or more electrical pulses or by calcium ionophore activation.

Nuclear addition comprises transfer of the donor nucleus into the recipient prior to enucleation. The donor nucleus is introduced into the recipient cell (in practice an oocyte, eg in GV, MI or MH phase), suitably by electrofusion or piezo-assisted micromanipulation. The resultant aneuploid cell is usually maintained to allow the donor nucleus to be reprogrammed before a reprogrammed diploid cell is generated, through loss, removal or destruction of its metaphase plate, nucleus, pronucleus, chromatin, chromosomes or DNA. In an exemplary technique, cytoplasm containing the metaphase plate is removed in the presence of cytochalasin B (Sigma, St Louis, MO). Before, concomitantly with or after transfer of the donor nucleus, the recipient cell is activated.

The donor nucleus may be of any suitable type and from any suitable species. The donor nucleus may be contained in a karyoplast or cell. The donor nucleus may be of embryonic, foetal, new born, juvenile or adult origin. Donor nuclei may be prepared by removing the nucleus and a portion of the cytoplasm and plasma membrane surrounding the donor nucleus from a suitable donor cell for example using microsurgery. Adult cells such as fibroblasts, cumulus cells, lymphocytes and neural cell types may be used. In a preferred embodiment, the donor nucleus may be from a somatic cell, more preferably an adult somatic cell, or a somatic stem cell. Cell lines may be used. In a particularly preferred embodiment embryonic cells such as embryonic stem cells or other pluripotent stem cell lines, embryonic germ cells or primordial germ cells may be used.

It is particularly preferred that the donor cells be at a particular stage in the cell cycle, for example G₀, Gi or S-phase. It is possible to isolate populations of cells which are enriched for cells at each stage in the cell cycle by sorting the cells on the basis of size, for example using FACS. This avoids the use of stains, which may be toxic to the cells. Staining can be used on a sample of each size-sorted population to identify what stage in the cell cycle that population is at.

The donor nucleus may be from a normal, abnormal .or genetically modified cell. Donor nuclei may be isolated directly from an animal cell, from an animal cell culture or from an established cell line. Abnormal cells include cells with known or unknown genetic mutations including gene and other chromosomal mutations, deletions, rearrangements, substitutions and/or duplications.

The recipient cell may be of any suitable type and from any suitable species. The recipient cell may be an in vivo or in vitro produced oocyte. Oocytes may be, for example, germinal vesicle (GV) stage or metaphase I (MI) immature oocytes, or oocytes arrested in the second metaphase of meiotic maturation (Mil oocytes). Other sources of recipient cells include zygotes, fertilised oocytes, embryonic blastomeres (eg 2-cell blastomeres) and cell lines produced from gonads, germ cell tumours or any other cell type suitable for allowing the succcessful addition of a nucleus. In addition, the recipient cell may be a pluripotent cell such as an embryonic stem (ES) cell, embryonic germ cell or primordial germ cell [which includes cells isolated from cell lines, primary cultures or isolated cells from the inner cell mass (ICM), embryonic disc (ED), embryonic ectoderm or primordial germ cells PCSs)], a cell derived from a tumor including an embryonal tumor may be used (eg embryonal carcinoma (EC) or yolk sac tumor cells). Somatic cell recipients such as neural or haematoietic stem cells may be used as a source of recipient cell. Oocytes, for example arrested in the second metaphase of meiotic maturation (MH oocytes), are preferred. Enucleation is described in more detail below under the heading "Enucleation of cells to yield 'karyoplasts' and 'cytoplasts'". Nuclear transfer is described in more detail below under the heading "Transfer of the donor nucleus to an oocyte".

In modifications of the Dolly and nuclear addition technologies, the donor nucleus is from a haematopoietic stem cell (HSC), as described in PCT/GBO 1/04229. The invention includes methods in which:

(i) nuclear transfer does use an HSC donor;

(ii) nuclear transfer does not use an HSC donor; (iii) nuclear transfer is by nuclear addition and does use an HSC donor; (iv) nuclear transfer is by Dolly technology and does not use an HSC donor.

In a preferred method of the invention, the donor cell is autologous.

In an alternative method of the invention, the donor cell is allogenic.

The terms autologous and allogenic are well known in the art. For the sake of clarity, autologous refers to a cell obtained from an individual, the nucleus of which is to be used for nuclear transplantation and subsequent cell mass and teratoma development. Allogenic refers to a cell obtained from another individual, the nucleus of which is to be used in the above procedure.

As already described, the donor cell may be a quiescent cell selected from any type of quiescent somatic cell (e.g. fibroblast) or it may be non-quiescent and preferably selected from haematopoietic stem cells and/or lymphopoietic stem cells. The use of a haematopoietic stem cell ('HSC') as donor is described further below under the heading 'HSC as donor'.

It will be apparent that haemopoietic stem cells (HSC with a CD34+, Lin -phenotype) can be isolated from either autologous cord blood or bone marrow/peripheral adult blood (see below under the heading 'HSC as donor' for more information). Non quiescent HSC's have advantages over quiescent somatic cells. For example they maintain an undifferentiated state, express high levels of telomerase, DNA remains in an unmethylated state maintaining the proliferative and developmental potential. Furthermore the autologous tissues when implanted will not be subjected to immune rejection therefore removing the need to administer immunosuppressive drugs.

Methods to grow haematopoietic stem cells are known in the art. For example, US5728581 describes bioreactors for expanding haematopoietic stem cells which involves the provision of culture conditions which promote the expansion of stem cells by combining variations in oxygen tension and addition of at least one cytokine which stimulates proliferation.

Collection of neonatal blood and selection of HSC

Hematopoietic cells (essentially CD34+ Lin- ) are isolated from neonatal blood contained in the cord placenta. The cells can be obtained easily and without trauma to the donor. US5004681, incorporated herein by reference, describes methods to obtain hematopoietic stem cells from the umbilical cord blood by immediate cord clamping after delivery and direct drainage. Volumes of 50 ml or more of neonatal blood have been obtained and found to contain enough of the appropriate cells to repopulate the entire hematopoietic system of an adult with an appropriate weight.

The method of the present invention provides for autologous (self) reconstitution and allogeneic cell/tissue transplantation whereby the nucleus of the hematopoietic stem cell is derived from another individual. Although autologous (self) reconstitution eliminates all risk of immune rejection, it will be apparent that this route will be most useful in the repair of injury, trauma or critical degeneration such as damage to the spinal cord, PD, CNS degenerative conditions, MI, diabetes, bone reconstitution. The allogeneic route may be preferred in circumstances where an individual has a genetic predisposition to disease or known genetic conditions impeding the autologous route.

Cryopreservation

Procedures and considerations for the manipulation, cryopreservation and long term storage of hematopoietic stem cells are well known in the art. Whole neonatal blood, as collected, is cryogenically frozen using known techniques.

Standard cell separation procedures and in-vitro stem and progenitor cell expansion are preferably carried out before cryopreservation to reduce sample volume and increase cell count respectively. In one embodiment of the invention, HSC cells are separated from other blood components and cryopreserved. In an alternative embodiment, the hematopoietic stem cells may be enucleated before cryopreservation and only the nuclei (nucleoplasts or karioplasts) cryopreserved under suitable conditions. Cryopreserving nucleoplasts instead of the entire cell, will cause a lower "cryogenic shock" due to their inherent lower water content (virtually no formation of water microcrystals) and lower concentration of cryopreservants. Furthermore, the absence of mitochondrial structures, which are abundant in the whole cell, will result in a lower oxidative stress which is a negative effect during the thawing out. In another embodiment of the invention, a reconstituted oocyte containing a HSC stem cell nucleus is cryopreserved. EP 0343217 describes methods for recovering stem and progenitor cells from the frozen state. Accordingly, cryopreservation of the HSC nuclei provides a stem cell bank which can be tapped when and if needed to provide a HSC nucleus for use in nuclear transfer into an enucleated oocyte. In an alternative embodiment of the invention, the HSC nucleus is transferred into an intact oocyte, following which the oocyte nucleus is removed.

Enucleation of cells to yield 'karyoplasts' and 'cytoplasts'

Selected treatment of cells in culture can result in cells extruding nuclei, resulting in the formation of separate nuclear and cytoplasmic parts named karyoplasts and cytoplasts respectively. In a preferred embodiment of the present invention, the recipient is enucleate. Enucleation may be achieved physically, by removal of the nucleus, pro-nuclei or metaphase plate (depending on the recipient cell). It may also be achieved functionally, such as by the application of ultraviolet radiation or another enucleating influence.

Different procedures have been used in attempts to remove the chromosomes with a minimum of cytoplasm. Aspiration of the first polar body and neighbouring cytoplasm was found to remove the metaphase II apparatus in 67% of sheep oocytes (Smith & Wilmut Biol. Reprod. 40 1027-1035 (1989)). Only with the use of DNA-specific fluorochrome (Hoechst 33342) was a method provided by which enucleation would be guaranteed with the minimum reduction in cytoplasmic volume (Tsunoda et al., J. Reprod. Fertil. 82 173 (1988)). In livestock species, this is probably the method of routine use at present (Prather & First J. Reprod. Fertil. Suppl. 41 125 (1990), Westhusin et al., Biol. Reprod. (Suppl.) 42 176 (1990)).

There have been very few reports of nαn-invasive approaches to enucleation in mammals, whereas in amphibians, irradiation with ultraviolet light is used as a routine procedure (Gurdon Q. J. Microsc. Soc. 101 299-311 (I960)). There are no detailed reports of the use of this approach in mammals. During the use of DNA-specific fluorochrome however, it was noted that exposure of mouse oocytes to ultraviolet light for more than 30 seconds reduced the developmental potential of the cell (Tsunoda et al., J Reprod. Fertil. 82 173 (1988)).

Enucleation results in a cell lacking a nucleus, but is otherwise intact for a number of days (Goldman et al 1973) These enucleated cells have been called anucleate cells (Poste 1972) or cytoplasts (Veomett et al 1974). The nucleus that is extruded from the cell retains a thin rim of cytoplasm and is surrounded by a plasma membrane; these structures have been called 'karyoplasts' (Veomett et al 1974) or 'mini-cells' (Ege and Ringertz 1975).

The nucleus of the hematopoietic stem cell may be separated by exposure to a pharmacologically effective amount of cytochalasin B. Cytochalasin B is an example of a chemical which is effective at separating the nucleus of a cell from the cytoplasm to form a karyoplast and cytoplast respectively, (Methods in Enzymology Vol 151, p221-237 1987). In a well-established technique, cells attached to a plastic disc are inverted over a solution of cytochalasin B in a centrifuge tube and centrifuged The cytoplasts remain attached to the plastic disc, while. the karyoplasts are pelleted at the bottom of the centrifuge tube (Prescott et al 1972). Alternatively, cells in suspension may be centrifuged through a density gradient, typically composed of Ficoll, containing cytochalasin B (Wigler and Weinstein 1975). In this case, cytoplasts and karyoplasts are formed and may be recovered from different parts of the gradient after centrifugation.

Any of the above techniques may be used to generate a donor nucleus which is preferably derived from a hematopoietic stem cell and a recipient oocyte cytoplast.

In one embodiment of the invention, the donor cell and recipient oocyte are of human origin. In an alternative embodiment, the donor cell and recipient oocyte are of non-human mammalian origin.

Transfer of the donor nucleus to an oocyte

The recipient cell to which the nucleus from the donor cell is transferred is in practice an oocyte. In a preferred embodiment of the invention, an HSC nucleus is transferred into an enucleated oocyte obtained by any of the above described techniques. In an alternative embodiment of the invention, the HSC nucleus is transferred into an intact oocyte. The oocyte nucleus is then removed accordingly. The oocyte is of human or mammalian origin.

It is preferred that the recipient host cell to which the donor cell nucleus is transferred is an enucleated metaphase π oocyte, an enucleated unactivated oocyte or an enucleated preactivated oocyte. At least where the recipient is an enucleated metaphase π oocyte, activation may take place at the time of transfer. Alternatively, at least where the recipient is an enucleated unactivated metaphase π oocyte, activation may take place subsequently. Three suitable cytoplast (enucleated oocyte) recipients are:

1. The "MAGIC Recipient" (Metaphase Arrested G1/G0 Accepting Cytoplast) described in WO9707668.

2. The "GOAT" (GO/G1 Activation and Transfer) - a MH (metaphase II) oocyte at the time of activation (Cambell et al., Biol. Reprod. 49 933-942 (1993).

3. The "Universal Recipient" (Campbell et al., Biol. Reprod. 649 933-942 (1993), Biol.

Reprod. 50 1385-1393 (1994).

All the above publications are included herein by reference.

Once suitable donor and oocyte cells have been identified, it is necessary for the nucleus of the former to be transferred to the latter. Nuclear transfer may be effected by fusion or micromanipulation.

Three established methods which have been used to induce fusion are:

(1) exposure of cells to fusion-promoting chemicals, such as polyethylene glycol;

(2) the use of inactivated virus, such as Sendai virus; and

(3) the use of electrical stimulation.

Methods for creating hybrid cells by fusing two or more cells of different origins together have been well documented. Kennett 1979 provides a review of the commonly used methods based upon Sendai virus induced cell fusion, or cell fusion induced by polyethylene glycol (PEG).

Cells to be fused are incubated with a fusogenic agent, such as Sendai virus or PEG. Centrifugation or agitation may be used to encourage clumping and close apposition of the cell membranes. Variables such as time, temperature, cell concentration and fusogenic agent concentration are optimised for each cell combination. These techniques have been shown to allow fusion of cytoplasts prepared by cytochalasin B induced enucleation and with whole cells or karyoplasts, also derived by cytochalasin B induced enucleation (Poste and Reeve 1971; Ege and Ringertz 1975; Ege et al 1973, 1974; Veomett et al 1974; Wright and Hayflick 1975; Shay 1977)).

Electrofusion is another well established and widely used method for inducing cell fusion. This involves passing short electric pulses through mixtures of cells (Neil and Zimmermann 1993).

While cell-cell fusion is a preferred method of effecting nuclear transfer, it is not the only method that can be used. Other suitable techniques include microinjection (Ritchie and Campbell, J Reproduction and Fertility Abstract Series No. 15, p60).

During fertilisation there are repeated, transient increases in intracellular calcium concentration (Cutbertson & Cobbold Nature 316 541-542 (19185). Electrical pulses are believed to cause analogous increases in calcium concentration. There is evidence that the pattern of calcium transients varies with species and it is anticipated that the optimal pattern of electrical pulses will vary in a similar manner.

Exposure of rabbit oocytes to repeated electrical pulses reveals that selection of an appropriate series of pulses and control of Ca²⁺ is necessary to promote development of diploidized oocytes to mid-gestation (Ozil Development 109 117-127 (1990)). The interval between pulses in the rabbit is approximately 4 minutes (Ozil Development 109 117-127 (1990)), and in the mouse 10 to 20 minutes (Cutbertson & Cobbold Nature 316 541-542 (1985)). There are preliminary observations in the cow to suggest that the interval is approximately 20 to 30 minutes (Robl et al., in Symposium on cloning Mammals by Nuclear Transplantation (Seidel ed.), Colorado

State University, 24-27 (1992)). In most published experiments activation was induced with a single electrical pulse. New observations suggest that the proportion of reconstituted embryos that develop is increased by exposure to several pulses (Collas & Robl Biol. Reprod. 43 877-

884 (1990)). In any individual case, routine adjustments may be made to optimise the number of pulses, field strength, pulse duration and calcium concentration of the medium.

2. Cell Division A pluripotent cell, for example an ES cell, EG cell or reprogrammed cell, is allowed to divide to allow the development of a mass containing one or more primary germ layer cells. Alternatively the original cell may be a totipotent cell.

Alternatively or additionally, the cell mass may be split and the cells clonally expanded to improve yield. Accordingly, the method of the present invention provides means for the preparation of a new cell-line which may act as a source of nuclear donor cells which can be produced from a cell mass formed according to the preceding description. Increased yields of cells may alternatively or additionally be obtained by clonal expansion of donors and/or by use of the process of serial (nuclear) transfer.

The cell mass derived by nuclear transfer is not an embryo (special culture conditions would normally be required in vivo). The developing cell mass undergoes development to give rise to a mass which comprises the three primary germ layers (ectoderm, endoderm and mesoderm) from which a cell or cell-line is derived.

The invention is not restricted as to the cell division process. Typically, it involves a period of division in host tissue, either in vivo or ex vivo. Often, but not always, the cell is allowed to divide in vitro to form a 2, 4, 6 or 8 cell mass prior to transfer to host tissue.

In vitro culture may be performed using standard techniques, including those used in INF (in vitro fertilisation). Commercial INF culture media may be used, for example those sold under the trade marks "IVF-20/-100" and "G-I" by Vitrolife Sweden AB, Mδlndalsvδgen 30, SE- 41263 Goteborg, Sweden. Alternatively, there may be used human tubal fluid, typically supplemented with maternal serum. Optionally, the pluripotent cells are cultured on a layer of oviductal cells, eg bovine oviductal cells.

Suitable culture conditions are 37°C under an atmosphere of 5% carbon dioxide.

Typically, in vitro culture is maintained until the pluripotent cell has divided to form a 6-8 cell mass, which is then implanted into host tissue. Alternatively, the 6-8 cell mass may be cultured in vitro, typically on a layer of oviductal cells.

A suitable host tissue is ex vivo; ideally it is tissue of the patient to be treated, eg muscle tissue, and the implanted cell mass is autologous to the patient. A plurality of cell masses may be implanted into one patient or host tissue mass. Where the host tissue is part of a male patient hormonal modification is usually required to reduce or block androgen production or activity and/or increase the level of oestrogens; such hormonal modifications may be local or systemic.

Ex vivo host tissue is suitably muscle strip, which optionally is modified by the addition of one or more factors, for example oestrogen, to promote maintenance of the introduced cell mass and differentiation into^" primary germ layer cells. Nutrients may be provided, for example, salts, glucose and amino acids.

Especially where the cell mass is transplanted into a recipient human or non-human animal, the cell-mass is optionally encapsulated in a biocompatible carrier. It will be apparent to one skilled in the art that the biocompatible carrier serves to retain the cells to prevent migration from the site of transplantation and thus to prevent the spreading of cells. However the carrier will be such that the diffusion of nutrients/ growth factors to the growing teratoma is not impeded. The fact that only a limited period of time is allowed in-vivo, further avoids the risk of neoplastic transformation.

Biocompatible carriers are known in the art. For example, US5976780, which is included herein by reference, describes an encapsulation device for cells for use in tissue transplantation or tissue implantation. The device is manufactured from porous sodium alginate and polysulfone fibres which facilitate the diffusion of nutrients required to maintain cells is a functional state.

The recipient may be an immunosuppressed selected animal model (syngenic, transgenic or cloned/transgenic animal suitable for xenotransplantation).

3. Separation of Primary Germ Cell

The cell mass containing primary germ cells is processed to separate out primary germ layer cells of different types using established histochemical techniques.

In preferred methods the cell mass is allowed to grow in a recipient animal or ex vivo tissue (e.g. segregated human tissue compartment) and excised from the recipient or tissue. 4. Implantation

One or more primary germ layer cells of one of the three types are surgically introduced into damaged or diseased tissue derived from the same type of primary germ layer cells, and allowed to differentiate to populate the tissue with healthy cells. The introduced cells are derived directly from a cell mass as described above,, or from a cell line established from one or more cells separated from a cell mass.

In other embodiments, genetically modified cells are introduced.

Nature control factors will help control division and differentiation of the introduced cells to populate the selected target tissue with appropriate differentiated cells.

5. Summary

The invention provides a process for preparing primary germ layer cells, isolating cells of each type and introducing them into a patient to differentiate. The invention includes:

• each stage of the process

• combinations of two or more sequential stages of the process • the product of each stage of the process and all intermediate products.

According to a yet further aspect of the invention there is provided a cell mass according to the invention encapsulated in a biocompatible material (carrier); the use of animal models (syngenic, transgenic or cloned/transgenic suitable for xenotransplantation) as temporary recipients as described; the use of segregated human tissue compartments as described.

According to a further aspect of the invention, there is provided a method for treating an animal or human by tissue engineering comprising surgically administering to a patient to be treated a primary germ cell.

Some examples of the therapeutic applications and value of this approach are the establishment, through the above technology, of autologous dopaminergic neurons or pre-neurons to be used for neurosurgical transplantation into the degenerated substantia nigra of patients affected by Parkinson's disease. Parkinson's disease is the result of progressive degeneration of dopaminergic neurons within the putamen area of the CNS. It has been already shown that neurons obtained from foetal brains, when surgically implanted, can restore the dopaminergic circuitry and have substantial long term clinical benefit in very advanced Parkinsonians. The strong limits of this therapeutic intervention are either the source of neurons and the immunological barrier (allogeneic neurons). This invention overcomes those limits entirely. Somatic quiescent cells (e.g. skin fibroblasts) or non-quiescent cells (e.g. haematopoietic stem cells such as CD34+ cells) can be easily and safely taken from a given Parkinson's patient, and they constitute the^' source of the nucleus. After the nucleus has been transferred into an enucleated oocyte (nuclear transfer technology), the reprogrammed cell divides to form a cell mass which, in some embodiments, is then implanted subcutaneously into the patient (other alternative methods of protected implantation or the use of surrogate hosts are described above) and allowed to growth for a limited period up to the formation of primary germ cells. After its excision, ectoderm-type cells are recovered for implanting into the diseased CNS area (Substantia Nigra).

The same applies for the transplantation of ectoderm-type cells to be used in patients with spinal cord injury for spinal cord repair and the replacement of CNS neurons and glial cells lost following ischemic and hemorrhagic stroke.

Another area of critical intervention is the establishment of autologous Langerhans beta-cells producing insulin or entire islets of Langerhans producing counteracting hormones such as glucagon, and their implantation in patients with severe and uncontrolled diabetes.

Yet another area of intervention is the establishment of striated myocardial cells by the injection, through coronary catheterism, of mesoderm cells into an acute area of myocardial infarction.

HSC as donor

According to this aspect of the present invention there is provided a method for forming a pluripotent cell, the method comprising transferring a hematopoietic stem cell nucleus into a recipient oocyte and, before or after transfer, removing the oocyte nucleus. The pluripotent cell is used for the manufacture of a cell mass comprising ectodermal, mesodermal or endodermal cells for use in implant/transplant therapy. In one embodiment of the invention, the donor cell and recipient oocyte are of human origin. In an alternative embodiment, the donor cell and recipient oocyte are of non-human mammalian origin.

Preferably the donor cell is cord blood haematopoietic stem cell.

Preferably the recipient oocyte is a metaphase II ^"oocyte, an unactivated oocyte or a preactivated oocyte.

Preferably the recipient oocyte is enucleated prior to transfer of the non-quiescent stem cell nucleus. In an alternative embodiment of the invention, the nucleus of the recipient oocyte is removed subsequent to transfer of the non-quiescent stem cell nucleus.

According to another embodiment of the invention there is provided a pluripotent cell comprising the nucleus of a hematopoietic stem cell and the cytoplasm of an enucleated oocyte.

According to one embodiment of the invention there is provided a pluripotent cell or a cell-line derived from any of the ectodermal, mesodermal and/or endodermal cells of a mass formed by the method.

Preferably the pluripotential characteristic includes the ability to proliferate in culture in an undifferentiated state.

In one embodiment of the invention the cell or cell-line has the capacity to proliferate in continuous culture in an undifferentiated state for at least 6 months and ideally 12 months.

Preferably the pluripotent characteristic includes the expression of at least one selected marker of pluripotent cells, for example a cell surface marker selected from the group comprising: SSEA-1 (1), SSEA-3 (+), SSEA-4 (+), TRA-1-60 (+), TRA-1-81 (+), alkaline phosphatase (+) and combinations thereof.

According to an alternative embodiment of the invention, there is provided a method for preparing a cell or cell-line comprising dissociating the cell mass formed by the above method to obtain dissociated cells. Usually the method further comprises growing the dissociated cells in culture under conditions conducive to proliferation of the cells and optionally storing the cells.

According to another embodiment of the invention, there is provided a method for inducing differentiation of at least one pluripotent cell comprising culturing the cell under conditions conducive to the differentiation of the cell into at least one tissue (e.g. neural-committed stem cells or glial-committed stem cells) and optionally storing the differentiated tissue.

According to yet another embodiment of the invention, there is provided a tissue or organ derived from a cell or cell-line of the invention selected form neuronal, muscle (smooth, striated and/or cardiac), bone, cartilage, liver, kidney, respiratory epithelium, spleen, skin, stomach, intestine and epithelial tissue. Preferably, the product comprising a suspension of committed cells, tissue or organ is combined with a suitable excipient, diluent or carrier and provided for use in tissue transplantation.

In one embodiment of the invention, ectodermal, mesodermal and/or endodermal cells derived from an HSC-derived mass of the invention and/or a pluripotent cell or cell-line derived from any of the ectodermal, mesodermal and/or endodermal cells of the blastula-type mass, are used for implant/transplant therapy by their direct introduction into the patient to be treated. In this embodiment, late-stage differentiation of the cell or cells into the required tissue type occurs in- vivo by orthotopic implant (i.e. ectodermal-derived stem cells into CNS/PNS).

According to a separate embodiment of the invention, there is provided a method to treat conditions or diseases requiring tissue and/or organ transplantation comprising providing at least one tissue type or organ according to the invention, optionally including a suitable excipient, diluent or carrier, introducing the tissue or organ into a patient to be treated and treating the patient under conditions which are conducive to the acceptance of transplanted tissue by the patient.

In one embodiment of the invention, the ectodermal, mesodermal, endodermal cell, cell-line, tissue or organ is autologous to the patient requiring treatment. Most preferably, the hematopoietic stem cell or the stem cell nucleus is obtained at birth of the subject and cryopreserved prior to use in the method. In an alternative embodiment, the ectodermal, mesodermal, endodermal cell, cell-line, tissue or organ is allogeneic to the patient requiring treatment. In one embodiment the hematopoietic stem cell or stem cell nucleus is cryopreserved prior to use in the method.

Contrary to GB 2318578 which teaches that the donor cell from which the nucleus is derived must be in the quiescent state, the present invention does not impose this pre-requisite upon selection of the donor cell and exploits the biological advantages of HSC nuclei for use in nuclear transfer. Advantageously, HSC nuclei maintain a more naϊve and plastic undifferentiated state and express the highest level of telomerase activity, together with DNA hypomethylation pattern. They thus maintain the developmental potential and indefinite proliferative capacity required to form adequate derivatives of all three embryonic germ layers and subsequent tissue-committed stem cells also in a proper quantity. Most kinds of cell therapy reconstitution in regenerative medicine usually require a proper amount of cells. In the example of neuronal-committed stem cells for treating Parkinson's disease, the amount of cells to be neurosurgically implanted through guided needle injections, might be in the magnitude of 10^Λ5 to 10^A8/site.

Nuclear transfer of a neonatal hematopoietic stem cell (HSC) nucleus into an enucleated donor oocyte or into an oocyte from which the oocyte nucleus is subsequently removed, and its in- vitro growth up to the Inner Cell Mass (ICM) and blastocyst stage provides the basis for the establishment of pluripotent cell-lines which, upon exposure to growth differentiation factors, can lead to the production of selected differentiated tissue for use, inter alia, in transplantation therapy. An example of growth/differentiating agents to be used for producing neuronal- committed stem cells are IGF-1, NGF, GDNF, retinoids and neurotransmitters (such as dopamine, serotonine, glutamate and others in various combinations).

The method of this aspect of the present invention allows for post-natal orthotopic (i.e. neural- committed stem cells injected into diseased CNS/PNS areas, or endocrino-committed stem cells injected into diseased endocrine organs) autologous (self) reconstitution or allogeneic cell/tissue transplant (donor to recipient) to achieve in vivo anatomo-functional reconstitution. In one embodiment of the invention, the human pluripotent cells and their tissue-committed derivatives can be used for post-natal autologous (self) reconstitution; the nucleus which is used for the nuclear transfer process is derived from a cryopreserved set of HSC of the same individual requiring the therapeutic intervention. Accordingly, no immune rejection is present and thus no immune suppression is required for a long lasting engraftment (the oocyte antigenic counterpart is diluted throughout divisions and will represent a minor antigenic pattern within the more differentiated cells).

It is well known that allogeneic material, if transplanted into another individual, may illicit a severe immune reaction in the host and thus be destroyed unless adequate, though toxic, immunosuppressive regimens are employed. The method of the present invention provides the important advantage that^", due to developmental^" immaturity and higher plasticity of the HSC, the material for use in transplant is less immunogenic and with greater proliferative nature. This provides for a greater rate of success with allogeneic cell/tissue transplant for which a certain critical number of committed stem cells might be warranted to obtain a successful tissue reconstitution. The allogeneic route may be preferred in circumstances where an individual has a genetic predisposition to disease and autologous (self) material cannot be used.

It will be apparent to one skilled in the art that the method of the present invention holds great potential for use in transplantation medicine, regenerative medicine, drug discovery and development and the study of human developmental biology.

It will be apparent that the methods described above for collection of neonatal blood and selection of HSC, cryopreservation, enucleation of cells to yield 'karyoplasts and cytoplasts' and transfer of a donor nucleus to an oocyte similarly apply.

In this embodiment a cell mass obtained as above is dissociated to obtain dissociated cells which are grown in suitable culture conditions. In one embodiment of the invention the culture conditions are conducive to proliferation and expansion of the cells. The cell culture may optionally be stored under suitable storage conditions.

In one embodiment, ectodermal, mesodermal and/or endodermal cells derived from a cell mass or a pluripotent cell or cell-line derived from any of the ectodermal, mesodermal and/or endodermal cells of the cell mass are used for implant/transplant therapy. Differentiation of the cell or cells into the required tissue type occurs in-vivo, providing that the appropriate layer cells are introduced into the right anatomical site (i.e. ectoderm cells or ectoderm-derived cells into CNS/PNS), thus avoiding the formation of a teratoma substrate.

Differentiation of at least one pluripotent cell of the invention may be induced by culturing the cell under conditions conducive to the differentiation of the cell into at least one tissue. In one embodiment of the invention, the differentiated tissue may be stored prior to use under suitable storage conditions.

A tissue or organ derived from a cell or cell-line of the invention may include neuronal, muscle (smooth, striated and/or cardiac), bone, cartilage, liver, kidney, respiratory epithelium, haematopoietic cell, spleen, skin, stomach, intestine tissue and endocrine tissue.

A tissue or organ obtained by the method of the invention may be combined with a suitable excipient, diluent or carrier and provided for use in tissue transplantation. The tissue or organ is introduced into a patient to be treated under conditions which are conducive to the acceptance of the transplanted tissue by the patient. Due to the pluripotent nature of a cluster of cells isolated from any of the germ layers, the autologous or allogeneic transplant should be done orthotopically (i.e. in the same anatomo-functional tissue sharing the same germ layer origin during embryogenesis). This will allow a further in-vivo growth and differentiation due to the presence of known and unknown tissue-specific growth/differentiating substances. This will also avoid a "reprogramming" path of the implanted pluripotent cells toward unwanted tissue types.

Claims

I . Use of a cell or a cell mass for the preparation of a primary germ layer cell for use in implant transplant therapy.

2. The use of claim 1 wherein the primary germ layer cell is an endoderm cell.

3. The use of -claim^" 1 wherein the primary germ layer cell is an ectoderm cell.

4. The use of claim 1 wherein the primary germ layer cell is an mesoderm cell.

5. The use of any preceding claim wherein the cell or cells is/are of human origin.

6. The use of any of claim 1 to 4 wherein the cell or cells is/are of non-human origin.

7. The use of any of any preceding claim wherein the cell or cells is/are obtained by transfer of a nucleus to a recipient cell.

8. The use of claim 7 wherein the nuclear transfer does not comprise transfer of a haematopoietic stem cell nucleus to an oocyte.

9. The use of claim 7 wherein the nuclear transfer does comprises transfer of a hematopoietic stem cell nucleus to an oocyte.

10. The use of any of claims 7 to 9 wherein the recipient cell is an enucleated oocyte.

I I . The use of any of claims 7 to 9 wherein the recipient oocyte is a metaphase π oocyte, an unactivated oocyte or a preactivated oocyte.

12. The use of claim 7 wherein the nuclear transfer comprises transfer of a hematopoietic stem cell nucleus to a recipient cell to produce an aneuploid cell, maintaining the aneuploid cell in a suitable environment for a period sufficient to allow the donor nucleus to be reprogrammed, and generating a reprogrammed diploid cell from said reprogrammed aneuploid cell by removal, destruction or loss of the recipient cell nucleus or nuclear DNA from said reprogrammed aneuploid cell.

13. The use of claim 7 wherein the donor nucleus is quiescent.

14. The use of any of claims 1 to 6 wherein the the cell or cells is/are obtained by in vitro fertilisation.

15. A method of providing a primary germ layer cell for use in implant/transplant therapy, comprising providing a cell mass comprising an endoderm cell, an ectoderm cell and/or a mesoderm cell, and separating from the cell mass a primary germ layer cell selected from endoderm, ectoderm and mesoderm cells.

16. A method of claim 15, wherein a plurality of cells of the same primary germ layer type are separated from the cell mass.

17. A method of claim 15 or claim 16, wherein the cell mass is prepared by

(i) transferring the nucleus of a somatic donor cell into a recipient enucleated oocyte;

(ii) activating the oocyte; and

(iii) providing conditions which are suitable for the division of the oocyte to a cell mass comprising cells of primary germ layer type.

18. A method of claim 15 or claim 16, wherein the cell mass is prepared by

(i) transferring the nucleus of a somatic donor cell into a recipient cell to produce an aneuploid cell;

(ii) maintaining the aneuploid cell in a suitable envirionment for a period sufficient to allow the donor nucleus tobe reprogrammed;

(iii) generating a reprogrammed diploid cell from said reprogrammed aneuploid cell by removal, destruction or loss of the recipient cell nucleusor nuclear DNA from said reprogrammed aneuploid cell; and (iv) providing conditions which are suitable for the division of the oocyte to a cell mass comprising cells of primary germ layer type.

19. A method of claim 17 or claim 18 wherein providing said conditions comprises introducing the oocyte or a cell mass derived therefrom into host tissue.

20. A method of any of claims 17 to 19 wherein the donor cell is quiescent.

21. A method of any of claims 17 to 19 wherein the donor cell is non-quiescent (e.g. haematopoietic stem cell from peripheral blood, bone marrow or from cryopreserved cord blood stem cells).

22. A method of any of claims 17 to 19 wherein the donor cell is not a haematopoietic stem cell.

23. A method of any of claims 17 to 19 wherein the donor cell is a haematopoietic stem cell.

24. A method of any of claims 15 to 23 wherein the cells of the cell mass are of human origin.

25. A method of any of claims 15 to 23 wherein the cells of the cell mass are of non-human origin.

26. A method of any of claims 15 to 25 wherein the cell mass is obtained by in vitro fertilisation.

27. A primary germ layer cell provided by a method of any of claim 15 to 26.

28. A primary germ layer cell or a cell-line derived from a primary germ layer cell provided by a method of any of claim 15 to 26.

29. A method to treat conditions or diseases potentially capable of benefiting from tissue and/or organ transplantation comprising introducing into a patient to be treated a primary germ layer cell of claim 27 or 28 and optionally a suitable excipient, diluent or carrier, the primary germ layer cell being introduced into tissue derived from primary germ layer cells of the same type as the introduced cells.

30. A method of implant/transplant therapy comprising performing a method of any of claims 15 to 26 and introducing into a patient to be treated a primary germ layer cell of claim 27 or 28 and optionally a suitable excipient, diluent or carrier, the primary germ layer cell being introduced into tissue derived from primary germ layer cells of the same type as the introduced cell.

31. A method of claim 29 or claim 30 wherein a hematopoietic stem cell is obtained at birth of the patient and cryopreserved prior to use of the cell as a nuclear donor in the preparation of the introduced primary germ layer cell.

32. A method of claim 29 or claim 30 wherein a hematopoietic stem cell nucleus is obtained from the patient and then cryopreserved prior to use of the nucleus in the method of the invention.

33. A method of claim 29 or claim 30 wherein the introduced primary germ layer cell is allogeneic to the patient.

34. A method of claim 29 or claim 30 wherein the introduced primary germ layer cell is autologous to the patient.

35. A method of generating a reprogrammed cell comprising:

(i) transferring the nucleus of a HSC donor cell into a recipient cell to produce an aneuploid cell;

(ii) maintaining the aneuploid cell in a suitable environment for a period sufficient to allow the donor nucleus to be reprogrammed;

(iii) generating a reprogrammed diploid cell from said reprogrammed aneuploid cell by removal, destruction or loss of the recipient cell nucleus or nuclear DNA from said reprogrammed aneuploid cell.

36. A method of producing primary germ layer cells, comprising providing a pluripotent cell and providing conditions which are suitable for the cell to divide to form a cell mass comprising primary germ layer cells.

37. A method of claim 36, wherein primary germ layer cells of each type are separated from the remaining cells of the mass.

38. A method of claim 36, wherein the pluripotent cell is initially cultured in vitro and then cultured in host tissue.