MXPA02010075A - Pluripotent cells comprising allogenic nucleus and mitochondria. - Google Patents

Abstract

Allochimeric ES cells are provided having nuclei from one individual of a species and mitochondria from a different individual of the same species. The ES cells can be used for differentiation into differentiated cell types for therapy or for studying various developmental processes in forming embryos and differentiation.

Description

PLURIPOTENT CELLS THAT COMPRISE ALOGENIC NUCLEI AND MITOCHONDRIA Field of the Invention The field of the invention is the manipulation of cells to provide non-human oocytes, for nuclear transfer procedures, wherein such nuclear transfer units possess allogenic nuclei and mitochondria from two different cellular sources of the same species. Background of the Invention The demonstration that nuclei can be transferred from a differentiated cell to an enucleated terminal cell, resulting in a dedifferentiated pluripotent cell, provides ample opportunities for biological manipulation, research and therapy. There is a growing opportunity to correct biological defects and adverse medical conditions, by using cells or organs that can replace or complement such a defect or condition. In the case of the transplant, up to now the trust in the organs obtained from donors has been had, where the donor is dead or there were two copies of the organ and the donor gave one of the copies. Despite efforts to encourage people to supply their organs for use after death, there remains a significant limitation of available organs and a broad waiting list. In those Ref: 142582 In cases where failure to replace the defective organ is fatal, there is a limited period of time in which a replacement organ is found. Not only are the organs in a limited supply, but the donor must be histocompatible with the recipient. Even with a close coupling of histocompatibility between the donor and the recipient, the recipient must normally be supported in immunosuppressive drugs to avoid rejection of the graft. These drugs often have serious adverse effects on the graft recipient, but they suffer because the graft loss would be fatal. As surgical capabilities have expanded, there is an increasing number of possibilities for cell and organ grafts to correct a large number of indications. Embryonic stem cells ("ES") offer opportunities to produce differentiated cells, organs and complete individuals upon request, so that one can clone a particular genotype. In the case of livestock animals, for example ungulates, the nuclei of similar embryos of preimplant cattle, support the development of enucleated oocytes until the end (Smith et al., Biol Reprod. 40: 1027-1035 (1989); Keefer, et al., Ibid 50: 935-939 (1994)). Due to the interest and importance of ES cells, there has been a large number of documents that they report various aspects of this technology. For example, Notarianni et al., J. Reprod. Fert. Suppl. 43: 255-260 (1991), reports the development of stable, pluripotent cell lines of pig and sheep blastocysts; Gerfen et al., Anim. Biotech 6: 1-14 (1995) report the isolation of embryonic cell lines from porcine blastocysts, whose cells differentiate into several different cell types during culture; Chemy et al., Theriogenology 41: 175 (1994) report cell lines derived from primordial germ cells of pluripotent bovine, maintained in long-term cultures, which formed embryoid bodies and which spontaneously differentiate into at least two different cell types; Campbell et al., Nature 380: 64-68 (1996) report the production of live lambs after the nuclear transfer of cultured embryonic disc cells ("ED") from day 9 sheep embryos cultured under conditions that promote isolation of the ES cell lines in the mouse; Van Stekelenburg-Hamers et al., Mol. Reprod. Dev. 40: 444-454 (1995) report the isolation and characterization of permanent cell lines from cells of an inner cell mass ("ICM") of bovine blastocysts, Smith et al., WO 94/24274, published October 7, 1994 and Wheeler et al., WO 94/26889, published on November 24, 1994, report the derivation of pluripotent ES cells of bovine and porcine, useful for the production of transgenic animals; Collas et al., Mol. Reprod. Dev. 38: 264-267 (1994) report the nuclear transplantation of cattle ICM by the microinjection of lysed donor cells into mature enucleated oocytes (see also, Keefer et al., Supra); Sims and collaborators, Proc. Natl. Acad. Sci. USA 90: 6143-6147 (1993) report the production of calves by the transfer of nuclei from ICM cells of bovine cultured in vitro in short term, in mature enucleated oocytes (see also, Stice et al., Biol. Reprod. : 100-110 (1996) Finally, Robl et al, PCT / US97 / 12919 report ES cells that result from a nuclear transplant between cross species, the extraordinary opportunities provided by the manipulation of ES cells, guarantee continuous research and the improvement in the character of the ES cells, the sources from which they can be derived, the way in which they expand and are carried to term, and the like.There is therefore a substantial interest in finding ways to allow the uses expanded of ES cells in the development of cells and organs or fragments of organs for transplantation, the breeding of domestic animals and the investigation of development processes that lead to the differentiation of cells and the formation of organoids, organs and portions thereof. Brief Description of the Invention Methods and compositions are provided that relate to modified oocytes and ES cells capable of expanding in culture and differentiating into different cell types, ES cells engineered, differentiated cells derived therefrom, organs or portions thereof. themselves and organisms derived from them. The methods comprise the use of an allogeneic embryo cell, wherein the allogeneic intends the final species pretended by the cell, which is enucleated and into which a xenogeneic nucleus is introduced, to provide a chimeric cell having a cytoplasm and mitochondria. of a species and a nucleus of a different species. After expansion of the chimeric cell to provide a nuclear transfer unit (NT) from which ES cells can be derived, ES cells can be used for further expansion and scientific investigation, or the NT unit can be transferred within of an allogeneic, syngeneic, or other compatible xenogeneic host uterus (based on the original oocyte) for pregnancy and birth. The oocytes of this animal Fl can then be harvested, enucleated and introduced into an allogeneic nucleus to provide an embryo cell that can supply cells that they have proteins of a simple species, that are immunologically competent and that can be accepted with a reduced risk of rejection when implanted in an allogeneic host. For the purposes of this application, these cells and their progeny will be referred to as "allochimeric". Of particular interest is the use of nuclei from a host who will be given differentiated allochimeric cells. Description of the Specific Modalities Allochimeric cells are supplied to understand the mitochondria of a first individual of a first species, and a nucleus of a second individual of the same first species. Broadly speaking, pluripotent cells capable of developing into a fetus or a plurality of different types of cells, will be produced by understanding mitochondria of an allogeneic species, wherein the allogenic species intends the species of interest, and a xenogeneic nucleus, wherein the genogenic nucleus claims the species in which the cells will be implanted or a different species compatible with the implant of the cells and the cells that differentiate into a fetus. The developed or mature fetus will then be used to harvest oocytes, enucleate the oocytes and introduce a nucleus of the same species as the mitochondria. The resulting allochimeric cells can be used in vitro and in vivo to study the differentiation, for produce cells along different differentiation trajectories, e.g., ectoderm, endoderm or mesoderm, and cells within these classifications such as neuronal cells, neurons, astrocytes, glial cells, ganglions, etc., hematopoietic cells, e.g., lymphocytes, macrophages, NK cells, erythrocytes, megakaryocytes, etc., fibroblasts, myoblasts, etc. The object methodology involves the following primary stages: 1. Harvest and enuclear embryo cells. 2. Introduce the nuclear genes of the mitochondria from a different species of embryonic cells in the nuclear genome of somatic cells of different species. 3. Introduce nuclei from a different species of the embryonic cells into the enucleated embryonic cells to provide chimeric cells. 4. Expand the chimeric cells to provide competent ES chimeric cells or incubation to develop nuclear transfer (NT) units. 5. I-nseminar the competent NT chimeric units in a compatible female host. 6. Allow the chimeric units to grow to a fetus, or finish and provide a newborn.

Grow in neonate up to an age to provide chimeric oocytes. 8 Harvest chimeric oocytes from the progeny. 9, Enucleating the chimeric oocytes and introducing allogenic nuclei to the mitochondria of the oocyte to provide allochimeric NT units. 10 Cultivate the allochimeric NT units to provide ES cells. 11. Optionally genetically modify allochemical ES cells. 12. Use the allochimeric ES cells to produce other cell types. Each of these stages in the process object will now be elaborated. 1. Harvest and Enucleation of Embryo Cells Embryo cells can be harvested from any convenient host of interest, for which the mitochondria of a species of interest is desired. Thus, any species of mammal of interest can serve as the source of the embryo cells, particularly domestic animals, more particularly large domestic animals, exemplified by equines, cattle, sheep, pigs, felines, canines, lagomorphs, murines, etc, and primates, exemplified by humans, monkeys and apes. Oocytes can be harvested in any convenient way, depending on whether they are sources of follicles of animals sacrificed or of which a surgical procedure is required. Of particular interest are primate cells, more particularly human cells that can serve for the production of differentiated cells. The cells can come from any member of the species, since the nucleus that will be used later will determine the species and the histocompatibility of the cells. The subject invention finds particular application with human cells, although there will be cases with other species wherein the allochimeric cells may play a role, eg, rare and valuable animals, animals for which in vitro fertilization is not reasonably feasible and the like. Human cells have been isolated and enucleated. In Zhang et al., J. Assist. Reprod. Genet 12: 361-8 (1995), the human fetal ovules are isolated and cryopreserved and upon thawing they are found to be able to mature until the formation of polar bodies. Follicular aspiration is described in Messinnis, et al., Br. J. Obstet. Gynaecol 93: 39-42 (1986); Pellicer, and collaborators, Hum. Reprod. 4: 536-40 (1989); Wahlstrom et al., Ann. N. Y. Acad. Sci. 442: 402-7 (1985); and Wood et al., Br. J. Obstet. Gynaecol 88: 756-60 (1981).

The techniques for enucleation follow the techniques used for other species and are described in a particular way in the Experimental section. The embryo cells can be matured in vitro in accordance with known techniques using a suitable maturation medium. Oocytes that have polar bodies (metaphase II oocytes) can be used as the host cells. See for example, Prather et al., Differentiation 48: 1-8 (1991) and Seshagine et al., Biol. Reprod. 40: 544-606 (1989)). Enucleation is achieved in accordance with conventional routes, conveniently using a micropipette to separate polar bodies and the surrounding cytoplasm. The oocytes are then separated to establish successful enucleation. 2. Introduction of nuclear genes of the mitrochondria from a different species of embryo cells within the nuclear genome of somatic cells of different species Somatic nuclei can be prepared by engineering, by introducing genes important for the function of the mitochondria in the type of mitochondria present in the embryo cell. The subject invention finds a particular application in the introduction of human genes of the nuclear mitochondria in the animal, particularly cells somatic of bovine to facilitate the cellular function of an animal cell with the human mitochondria. 3. Introduction of the nuclei of a different species of the embryo cells within the enucleated embryo cells to provide chimeric cells A wide variety of mammalian hosts can be employed as the source of the nuclei, where the question will be mainly one of convenience. In addition to the need to grow the chimeric NT unit until the end, other considerations in the selection of the host source to harvest the host oocytes, ease of handling and growth in the crop, maturity of the technology, rate of previous success in achieving progeny, ease of host growth, and ability of the resulting chimeric oocytes, number of available oocytes, compatibility of the mitochondria with the host nucleus, as well as other practical considerations. Although any mammalian host can be employed, domestic animals, more particularly animals or large domestic primates other than humans, are of particular interest. Exemplary mammals include ungulates, bovines and sheep, pigs, felines, equines, lagomorphs, murines, canines, etc. The isolated oocytes are matured in vitro and selected for the presence of polar bodies.

The nuclei can come from any source, somatic or germ cells, which can provide progeny production. Therefore, the nuclei may come from fetal cells, neonatal cells, mature cells, cells produced in a culture medium or the like. Generally, neoplastic cell nuclei will not be used, but may have application depending on the nature of the neoplasm. The source can be differentiated cells, which can be quiescent, G0 or in an active state, for example, Gi or G2. Cell types include embryos or pronuclei of a cell (zygotes), embryonic cells, blastomeres, epithelial cells, endothelial cells, muscle cells, keratinocytes, skin cells, alveolar cells, hepatocytes, renal cells, neuronal cells, cells hematopoietic, fibroblasts, endothelial cells, parenchymal cells, fat cells, glial cells, follicular cells, etc., where the choice can be determined by various factors associated with the final purpose. There are several techniques for introducing the nucleus into the enucleated embryo cell. Fusion and injection has proven to be efficient where the cell, which is the source of the nucleus, is placed in the space of the enucleated oocyte's perivitellin and fused in a fusion chamber by means of an electrical pulse or by using a Fine glass needle, the donor nucleus or the pronucleus of a fertilized oocyte can be isolated and delivered into the cytoplasm of the recipient embryo. These pronuclei may be from animals whose genomes have been modified to include nuclear genes from the mitochondria of the same species as the receptor oocyte (see, for example, Prather et al, U.S. Patent No. 4,997,384). The fusion can also be achieved by the use of electrical pulses or by the use of various fusogenic reagents, for example, the Sendai virus. (See, for example, Graham, Wister Inot, Symp Monogr. 9:19 (1969) and Collas and Barnes, Mol. Reprod Dev. 38: 264-267 (1994)). 4. Expansion of the chimeric cells to provide competent chimeric ES cells or incubation to develop nuclear transfer (NT) units. After fusion, the resulting fused nuclear transfer units ("NT") are placed in a suitable medium until the activation, for example, CRIaa. Typically, the activation will be made soon after, usually within 24 hours, more usually 4-9 hours later. Activation can be achieved by methods known to mammalian cells, such as the culture of the NT unit at a sub-physiological temperature, for example, room temperature, oocyte penetration by sperm, electrical and chemical shock, etc. See for example, Susko-Parrish and collaborators, patent E.U.A. No. 5,496,720. Other methods include making a simultaneous or sequential increase in the levels of divalent cations in the oocyte, for example, calcium, magnesium, barium, or strontium, conveniently in the form of an ionophore, by using electric shock, ethanol treatment and / or treatment with chelators. in cages; or reducing protein phosphorylation in the oocyte, using kinase inhibitors, for example, 6-dimethylaminopurine, staurosporine, roscovitine, butyrolactone, 2-aminopurine, and sphingosine, or by introducing one or more phosphatases into the oocyte, for example, phosphatase 2A or 2B. One technique of interest for activation is: activation of the NT units within 18 to 30 hours after the start of the culture in the maturation medium. The NT units are separated after the culture media and placed in activation media (2ml of Hepes TL with 2mg of bovine serum albumin, 5mM of Ionomicin and 2mM of 6-DMAP for 4 minutes in a sliding lid heater.) At the end of 4 minutes, the NT units are rinsed in Hepes TL and are placed in culture media with 2 mM of 6-DMAP at 38.5 ° and 5% C02 for 2 to 5 hours.At the end of this period, NT units are rinsed 4 times with Hepes TL and placed in culture. When the desired development stage is reached, the ES cells will be derived or the NT units will be transferred to the recipient females.

The activated NT units of chimeric cells can then be cultured in a suitable culture medium in vitro or in vivo, until the generation of ES cells and colonies of cells that develop embryos such as blastocysts. Suitable culture media for the culture and maturation of ES cells and embryos are well known in the art. Chimeric cells can be treated as cells of the core species, which have the surface membrane proteins, nuclear and cytoplasmic proteins defined by nuclear genes. Examples of known media include 10% fetal calf serum + Ham F-10 ("FCS"), tissue culture medium -199 ("TCM-199") + 10% fetal calf serum, tirodespalbúmina- lactate-pyruvate ("TALP"), buffered Dulbeco's phosphate buffered saline ("PBS"), Eagle's and Whitten's media. A common medium is TCM-199 or DMEM and 5 to 20% fetal calf serum or an equivalent source of growth-supporting factors such as newborn serum, heat-whey, lamb serum or beef serum. One exemplary medium is TCM-199 with Earl salts, 10% fetal calf serum, 0.2mM Na pyruvate and 50μg / ml gentamicin. These media can be used with cell lines to provide a conditioned medium, with the use of granulosa cells, oviduct cells, BRL cells, uterine cells and STO cells. Alternatively, the means described by Rosenkrans, jr. , Patent E.U.A. No. 5,096,822. This medium referred to as CR1 contains hemicalcium L-lactate in from about 1 to 10 mM, usually 1 to 5 mM, sodium chloride, potassium chloride, sodium bicarbonate, and a lower amount of acid-free bovine serum albumin. fatty, and when essential and non-essential amino acids are added, the medium is referred to as CRlaa. An exemplary CR1 medium comprises 114.7 mM NaCl, 3.1 mM KCl, 26.2 mM Na2C03, 5 mM Hemicalcium L-lactate and 3 mg / ml acid-free bovine serum albumin. Conveniently, activated NT unit chimeric ES cells are placed in a CRlaa medium containing 1.9 mM DMPA for about 4 hours, followed by a wash with HECM and then cultured in CRlaa containing BSA, at about 38.5 ° C, C02 at 5%, for about 4-5 hours. The NT units grown are usually washed and placed in an appropriate medium conditioned for growth. An effective medium is the CRlaa medium containing 10% FCS and 6 mg / ml BSA. Suitable feeder layers include fibroblasts and epithelial cells, particularly uterine epithelial cells, from sources such as ungulate, chicken, murine, STO, SI-m-220 and BRL cells. Mouse embryonic fibroblasts have been found to be particularly useful. After a sufficient time, which may vary with the species of the nucleus, chimeric ES cells are obtained which can now serve for insemination in an appropriate host. 5. Insemination of competent chimeric NT units in a compatible female host. 6. Permission to chimeric NT units to grow up to a fetus or to the end and provide a neonate. These stages are well established in the literature by a number of species, including murine and ungulate. For insemination, the NT units are transferred to the uteri. After insemination, the host is observed to ensure that NT units have been successfully implanted. Depending on the nature of the host, the host may be restricted to prevent a miscarriage. The delivery of fetuses or neonates can be a spontaneous or induced abortion, natural delivery or cesarean section. 7. Growth of the neonate up to an age to provide chimeric oocytes. . 8. Harvest of chimeric oocytes of the progeny. The above steps will depend on the nature of the host. The normal maintenance of the host is used to allow the neonate to grow as a healthy host. The harvest of the oocytes can be done as described above. 9. Enucleation of the chimeric oocytes and introduction of the allogenic nuclei to the mitochondria of the oocyte to provide the allochimeric NT units. The manner of enucleation and introduction of nuclei has already been discussed. The selection of the species to supply the nucleus will depend on the purpose for which the cells will be used. The cells can be used to clone a particular species, particularly where the oocytes are only difficult to grow in the species, such as humans or rare species, or as a source of differentiation cells, where the cells have both a nucleus and mitochondria of the same species. Unlike the human, another - a species that finds application with the subject invention includes cloning rare and endangered animals. 10. Cultivate allochimeric NT units to provide ES cells. NT units are grown in the layer, feeder until the NT units reach an appropriate size for the ES cell isolate, generally requires at least 4 cells, preferably at least 50 cells, and usually not exceeding 400 cells. The crop will usually employ conditions of 38.5 ° C and C0 at '5%, with the culture medium changed around every 1-5 days. The final stage involves mechanically removing the NT units from the culture, isolating the cells from the Within the NT unit, however, cells from other portions of the NT unit may find application, wash the cells of the NT unit and place the cells in a feeder layer, for example irradiated fibroblast cells, of the same or different species of the nucleus. The cells are maintained in the feeder layer in an appropriate growth medium, for example alpha-MEM supplemented with 10% FCS, β-mercaptoethanol and 0.1 M L-glutamine. The growth medium is conveniently changed around every 1-3 In the case of a human allochimeric ES cell, the individual cells are not well defined and the perimeter of the colony is refractive and uniform in appearance. The cells do not have an epithelial-like appearance. The NT unit cells can be used to provide internal cell mass (ICM) cells of blastocysts. ICM cells can grow in the culture using feeder layers, for example STO (mouse fibroblasts) with serum stripped with mineral carbon. 11. Optional genetic modification of allochimeric ES cells. _ ". ES cells can be modified with DNA according to conventional techniques. Uncovered DNA, DNA coated by recA, viruses, plasmids, YAC, extrachromosorna1 DNA, chromosomal fragments, cDNA or other DNA can be introduced. source of the desired gene, regulatory sequence or the like, in ES cells, with the use of an appropriate medium, liposomes, transporter sequences, permeation, electrofusion or the like. See, for example, Schneike et al., Science 278: 2130-3 (1997). Modifications can be provided for the constitutive or inducible expression of a particular product, for example insulin angiogenesis factors, cytokines, for example interleukins, interferons and colony stimulating factors, blood factors, for example serum albumin, thrombin, fibrinogen, thrombopoietin, erythropoietin, tissue plasminogen activator, etc., hormones, for example growth hormones, growth factors, for example epidermal growth factor, basic fibroblast growth factor, glial-derived neurotrophic growth factor, etc., enzymes, enzyme inhibitors, for example a-antitrypsin, proteins associated with telomere, for example Sir, telomerase, etc., neuronal proteins, for example neutrofin-3, 4/5, ciliary neutrophic factor, etc., pancreatic proteins, basal proteins, proteins of taste primordium, eye proteins, hemoglobin, transcription factors, lipoproteins, immunoglobulins, surface membrane receptors, for example insulin receptor, oncogenic repressors, L-dopamine, and angiogenesis inhibitors. Alternatively, to correct an undesirable defect or phenotype, homologous recombination in the nucleus which will be used to produce the allochimeric ES cells can be used. For example, the nuclei of subjects suffering from sickle cell anemia can be genetically modified to provide cells that provide a native hemoglobin; subjects suffering from hemophilia may have the corrected blood factor mutant gene, for example Factor VIIIc or VIIIvw, Christmas factor; subjects susceptible to diseases due to genetic factors, may have their nuclei modified to provide alleles less susceptible to such diseases, for example AIDS, juvenile onset diabetes, muscle atrophy, Alzheimer's disease, Parkinson's disease, and others.

Instead of introducing a genetic capacity, in some situations it may be desirable to turn off a genetic capacity or make the gene inducible. Illustrative genes that can be put out of circulation include oncogenes, histocompatible proteins, blood type proteins and other genes whose dominant expression leads to pathology. Homologous recombination or separation by exclusion cells can be used in which the desired agent is present. 12. Use of allochimeric ES cells to produce other cell types.

Pedersen, Reprod. Fertile Dev. 6: 543-52 (1994) reviews a number of articles concerning the differentiation of ES cells. The author reports the production of differentiated cells that express the specific CD markers for the type of cell and stage of differentiation or products produced by the cells, whose products are normally produced by the cells at the level of differentiation of the ES cells. The development of ES cells in specific cell types can be found in Bain et al., Dev. Biol. 168: 342-357 (1995) (neural cells); Palacios y Colaboradores, Proc. Natl. Acad. Sci. USA 92: 7530-7537 (1995) (hematopoietic cells); and Rathjen et al., Reprod. Fertile. Dev. 10: 31-47 (1998). Cells of interest include hematopoietic cells, neuronal cells, skeletal and cardiac muscle cells, skin cells, epithelial cells, endothelial cells, structural cells, osteoclasts and osteoblasts, follicular cells, eye cells, cells associated with sensitivity, such as cells of the inner ear of the primordial taste, bone cells, renal cells, hepatocytes, pancreatic cells, for example ß-islet cells, fibroblasts, and chondrocytes. Differentiated ES cells can find application in a wide variety of jurisdictions. The cells have allogeneic mitochondria and cells that provide nuclei that represent normal cells comprising DNA of a single species. In this way, the questions concerning the incompatibility between the mitochondria and the nucleus are obvious, the proteins that appear in the cytoplasm are all of the same species, the interaction between the nucleus and the mitochondria, for example the transport of proteins is natural, and the immunodominant sequences present on the surface of the cell will all form the same species. Additionally, cellular processes that depend on the interaction between the mitochondria and other intracellular compartments would also be natural. ES cells and differentiation cells offer many opportunities for the dissection of cellular processes, response to exogenous agents, for example genes, drugs, factors, etc., used to separate the effect of these exogenous agents in the development of drugs, identify specific alleles or mutations associated with the response to these external agents, and investigate variations and expression in response to various agents. In addition, because the ES cells can be maintained in culture and expanded, differentiated cells can be produced repetitively, where the cells are based on the same genotype. The subject invention also offers the ability to use the core of an individual for research and diagnosis. In this way, the susceptibility of a particular individual to external agents, such as carcinogens, allergens, toxins, and mutagens where there is a complete cell of a simple species that has the native metabolism of that species. The cellular response to a particular regimen, for example drug regimen, can also be investigated to determine the effect of normal cells of a particular patient. For chronic administration of a regimen, there is a sufficient time to use the enucleated stored ovum of the species of interest as a receptacle for the introduction of nuclei of differentiating cells of a particular individual of such a species. The ES cells are then able to be directed to produce various differentiated cells at different stages of differentiation, which can be subjected to the regime and changes in the character of the determined cells. There will be situations where the invention object allows to study the processes of the mitochondria in conjunction with a particular nucleus. For example, where an individual may have a nucleus that is defective to provide proteins that are used by the mitochondria in this process, the subject methodology allows the cloning of such cells and foresee the effect of differentiation on the mitochondrial process and the effect in growth patterns, phenotype, etc.

In those instances where the ES cells or the NT units are used for the maturation of a fetus and the production of a neonate, the use of the native mitochondria avoids the incompatibility when the resulting individual is mated with other individuals of the same species. In this way, rejection of the fetus by the mother is less likely and immune responses to the host cells are less likely, as a result of the presence of foreign mitochondrial proteins. The compositions which find use in the subject invention are oocytes comprising mitochondria of a species and a nucleus of a different species, isolated and in culture. For the most part, the nucleus and the mitochondria will be of species that are substantially different, generally of different genera and even of different families. Conveniently, the nucleus will be chosen to provide a useful source of the enucleated ovule, such as from domestic animals (ungulates), for example bovine, ovine and porcine, and from laboratory animals, for example murine. Also included in the subject invention are the ovules comprising mitochondria of an individual and a nucleus of a different individual of the same species, the ES cells are derived from these, the cultures comprising such ES cells, differentiated cells derived therefrom, and cultures comprising such differentiated cells.

The compositions include allochimeric oocytes and ES cells in growth cultures for cell expansion. The compositions also include mixtures of allochimeric cells and cells differentiated from internal cell masses or concomitant growth or maintenance of ES cells and differentiated cells in the culture medium for cell differentiation. For hematopoietic cells, a culture medium comprising monolayers of bone marrow stromal cells RP0.10 treated with mitomycin C (5-10 μg / ml at 37 ° C for 3-4 hours) or irradiated (2-4 x 103 radiations of rays ? 1 radiation = 0.01 G?) In plates of six Costar wells, in the presence of recombinant interleukin-3 (rIL-3) (100-300 units / ml), rIL-6 and F (final concentration 10% v / v ) of 2 day culture supernatants of confluent FLS4.1 fetal liver stromal cells. The FLS4.1 supernatant contains the ligand FLT3, steel factor and a new factor that supports the growth of hematopoietic stem cells in 2-2.5 ml of culture medium [Iscove Dulbecco modified medium / 50μM 2-mercaptoethanol / 2mM L-glutamine / gentamicin at 50μg / ml / 7.5% FCS v / v] at 37 ° C in an atmosphere of 7.5% C02 / 92.5% air. Every 5-7 days, the cells were harvested and subcultured in plates of 6 new Costar wells containing stromal cells R.P.0.10 treated with mitomycin.

C and culture medium supplemented with freshly prepared cytokine. (Palacios, et al, Proc. Natl. Acad. Sci. USA 92: 7530 (1995)). It will be understood that other cell lines that provide an analogous conditioned medium can be used, as well as other constituents that provide analogous activities. For neuronal cells, ES cells are subjected to an induction procedure of 8 days, consisting of 4 days of culture as aggregates without retinoic acid (RA) followed by 4 days of culture in the presence of RA. The medium is DMEM (high glucose with L-glutamine, without pyruvate, GIBCO 11965-043), 10% fetal bovine serum, 10% newborn calf serum and nucleoside reserve solution. Other means can be employed for targeted differentiation in other cell types, with the use of factors that naturally exist and affect differentiation in vivo, such as hematopoietic factors, for example G-CSF, M-CSF, GM-CSF, interleukins, interferons and other cytokines and growth factors. The enucleated oocytes, all having the same mitochondria, can be stored frozen in an appropriate medium for prolonged periods and then thawed carefully before use. Differentiated cells, whether genetically modified or not, find use in therapy, where Healthy cells can be introduced into the appropriate compartment to perform a desired function. For example, myocytes can be used to graft to myocardial tissue or other muscle tissue, where natural or genetically modified cells can provide an advantage, for example the correction of a mutant protein, as in the case of muscle atrophy. See, for example, Patent E.U.A. 5,602,301. Hematopoietic cells can be infused into a patient, such as lymphocytes, natural killer cells, megakaryocytes, eosinophils, basophils, mononuclear cells, or precursor cells, where the precursors can be dedicated to a specific cell type or they can be multipotent and differentiate various different types of cells. The ß-islet cells can be grafted to the pancreas or provided in a protective container that can be innervated by a blood supply for the treatment of diabetes. Neuronal cells can be introduced into the cerebral cavity to provide therapies for Parkinson's disease, Alzheimer's disease, cerebral palsy, and the like, by providing a source of L-dopamine, NGE or other factors or other composition that can affect brain function. Other indications for the use of ES object cells or their differentiated progeny include spine lesions, multiple sclerosis, liver diseases, vascular diseases, replacement of burned cartilage, heart disease, kidney disease, urinary tract diseases, prostate diseases, and diseases that result from aging. The subject cells can be used to provide increased supplies of particular factors, constitutively or inducibly. Pruschy et al., Chem. Biol. 1: 163-72 (1997) provide modifications that allow the induced production of a product by orally taking a pill, which activates a transcription factor. In this way, the production of activating plasminogen tissue can be induced in the case of heart attack, insulin production by diabetics, activation of lymphocytes in case of infection, during the introduction of non-human receptors such as the ecdysone receptor, and the like. The following examples are by way of illustration and by way of limitation. EXPERIMENTAL Materials and Methods Human receptor oocytes: Oocytes of human fetuses: Fetal ovaries are obtained from human fetuses 16-20 weeks of gestation, after a selective abortion. Ovarian tissues were shredded to ~ lmm in size and cultured in medium supplemented with 15% (v / v) fetal bovine serum, 0.13 IU / ml FSH and 35 ng / ml insulin. The tissue was cultured at 37 ° C in 5% C02 air for 5-25 days in Falcon dishes and 3-40 days in Costar Transwell-COL membranes before the induction of final maturation in the presence of LH and human follicular fluid. . The monolayer fragments consisting of fibrolasts are formed within 2-3 days of fresh tissue culture. After 1 week of culture, the follicles separate from the ovarian tissue, but remain bound to the monolayer. The maximum number of follicles that separate from the tissue appears about 1 week after starting the culture. After 40 days of culture in the Costar dishes, a significant fraction of the ovule reaches a diameter of more than 80μ, of which around a third is surrounded by the zona pellucida. After the induction of the final maturation, the extrusion of the first polar body is noted in the ovary that grew in the Costar dishes for 40 days. The mature ovum can be collected and used directly for enucleation. Oocytes of mature human females: Human ovules are obtained with the use of the Trotnow et al. Technique, Arch. Gynecol. 236: 211-7 (1985). A steering attachment is used with the Diasonics DS 1 sector scanner transducer using a suction needle slid through a trocar, with a continuous sonographic image of the aspiration needle. The patient is given an epidural anesthetic.

Other techniques may be employed, including administering a follicle stimulating hormone in accordance with the regimen described by Mercan et al., Hum. Reprod. 12: 1886-9 (1997). The oocytes can then be matured in vitro as follows. The immature oocytes are washed in TL-HEPES buffer medium containing 3 mg / ml bovine serum albumin (Fraction V). The oocytes accumulated in complexes are placed in TCM-199 containing 10% fetal calf serum, LH and / or FSH and estradiol at 39 ° C. After about 20 hours in the maturation medium, the oocytes are removed and placed in TL-HEPES with 1 mg / ml of hyaluronidase and the accumulated cells are removed by repeated pipetting through a fine diameter pipette. The depleted oocytes were separated from the polar bodies and those containing polar bodies (metaphase II oocytes) were selected for further use. Donor cells of cattle: Heifers were artificially over-ovulated and inseminated. The embryos of the reproductive tract were collected on day 5 or 6 after estrus was established. The embryos were removed from the reproductive tract with PBS and harvested. Embryos matured in vitro, fertilized and cultured were used as donor embryos on day 5 of fertilization. The fertilization process is described in Keefer et al., Mol. Reprod. Dev. 36: 469-74 (1993).

Enucleation: Enucleation was performed with the use of a bevelled micropipette approximately 18 hours after the onset of maturation (hpm). Enucleation was confirmed in the TL-HEPES plus bis-benzimide medium (Hoechst 33342, 3 μg / ml). Nuclear transfer: Individual donor cells were placed in the perivitelline space of the receptor enucleated oocyte. The cytoplasm of the human oocyte and the bovine donor nucleus (NT unit) were fused together with the use of electrofusion: a fusion pulse of 90V for 15 μsec to 24 hours after the onset of oocyte maturation. The resulting NT units were placed in a CRlaa medium until 28 hours after the start of maturation, at which time they were activated in accordance with the following process. NT units were exposed for 4 minutes to ionomycin-6-DMAP (2mM) followed by 4 hours in DMAP alone (2mM) in TL-HEPES supplemented with 1 mg / ml serum albumin and then washed for 5 minutes in TL -HEPES supplemented with 30 mg / ml of serum albumin. The NT units are then transferred into a micro-drop of CRlaa culture medium plus FCS at 10% and 6 mg / ml of serum albumin in 4 wells of plates containing a confluent feeder layer of mouse embryonic fibroblasts. The NT units are they cultivate for 3 more days at 38.5 ° C and C02 at 5%. The culture medium was changed every 3 days until day 12 after the start of activation. At this time, about 50 cells should be formed, whose cells are mechanically removed from the area and used to produce embryonic cell lines. Feeding layer of mouse embryonic fibroblast: Primary cultures of mouse embryonic fibroblasts of murine fetuses 14-16 days old are obtained. After the head, liver, heart and alimentary tract were aseptically removed, the embryos were minced and incubated for 30 minutes at 37 ° C in pre-warmed EDTA trypsin solution (0.05% trypsin / 0.02% EDTA; , Grand Island, NY). Fibroblast cells were seeded into the tissue culture flasks and cultured in an alpha-MEM medium (BioWhittaker, Walkersville, MD) supplemented with 10% FCS, penicillin. (lOOIU / ml) and streptomycin (50μl / ml). Three to four days after passing, the embryonic fibroblasts were irradiated in 35 x 10 Nunc culture plates (Baxter Scientific, McGaw Park, IL). The irradiated fibroblasts were grown and maintained in a humidified atmosphere with 5% C02 in air at 37 ° C. Culture plates that have a uniform monolayer of cells are used to culture embryonic cell lines.

Production of embryonic cell lines: NT unit cells obtained as described above were washed and placed directly into cells of irradiated feeder fibroblasts (see above). These cells include those from the inner portion of the NT unit. The cells are maintained in a growth medium consisting of alpha-MEM supplemented with 10% FCS and 0. lmM of β-mercaptoethanol. The growth medium is exchanged every 2 to 3 days. The initial colony is observed by the second or third day of cultivation. The colony spreads and exhibits a morphology similar to the embryonic stem cells (ES) of cattle and mice previously described. The individual cells within the colony are not well defined and the perimeter of the colony is refractive and uniform in appearance. The cells have an epithelial appearance. Production of chimeric NT units of cattle: The chimeric NT units are selected and implanted in cows in accordance with the following procedure: 1-5 NT units are implanted in the uterus. The resulting calves grow to the appropriate stage to develop oocytes, at which time the ovules are aspirated and isolated. Alternatively, oocytes from adult females are obtained by over-ovulation and by oocyte-guided oocyte retrieval of sacrificed cows.

Production of human allochimeric cells: The chimeric ovules obtained as described above are treated as described above for growth and enucleation, and are then ready to be used for the reception of human nuclei. Human epithelial cells are gently scraped from inside the mouth of human adults with consent with a regular glass slide. The cells are washed completely from the slide in a Petri dish containing PBS with Ca or Mg. The cells are pipetted through a small diameter pipette to break up cell clusters in a single cell suspension. The cells are then transferred into a micro-drop of TL-HEPES medium containing 10% FCS under oil for the nuclear transfer of chimeric enucleated bovine oocytes comprising human mitochondria with the use of the procedure described above. Allochimeric cells can employ nuclei of substantially any convenient human cellular source, such as fibroblasts, epithelial cells, keratinocytes, blood lymphocytes or gland epithelial cells. The allochemical cells are grown in culture and directed in various differentiation patterns, as described by Stice et al. (1998), supra, which includes the preparation of myocytes.

In accordance with the subject invention, human allochimeric cells having human nuclei and human mitochondria of different individuals are provided. These cells find an extensive use in that they provide a simulated natural human cell to study the cellular processes in the development of embryos and the differentiation for differentiated types of cells. The cells can also be used to produce differentiated cells by cell therapy or human factor production. The target cells provide cells that produce only human proteins, as well as are less likely to induce an immune response, allow genetic manipulation of the nucleus, to provide human cells that can be introduced into an individual that is the source of the nuclei, but in where the nuclei are modified for therapeutic or other purposes, and provide a source of genotypically identical cells, which can be used to clone a particular genotype. The references cited in this application are incorporated herein for reference as they have been fully placed in the specification. All procedures and methods are included as if they were placed and were part of the procedures of this application, when applied to the subject matter of this application in accordance with those skilled in the art.

The invention that has now been fully described will be apparent to one of ordinary skill in the art that many changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (20)

1. Claims Having described the invention as above, the content of the following claims is claimed as property. A method for producing allochimeric cells comprising mitochondria of a first human and a nucleus of a second and different human, the method characterized in that it comprises: enucleating an oocyte of a first human; introducing the nucleus of a species other than human into the enucleated oocyte to provide a first chimeric oocyte; expanding the first chimeric cell in the culture to provide NT units; inseminating the ES cell in a compatible female host and allowing the NT units to grow to at least one fetus comprising the second chimeric oocytes; harvest and isolate at least one of the second chimeric oocytes; enucleating at least one of the second chimeric oocytes and introducing a human nucleus into the second chimeric oocyte to provide an allochimeric NT unit; and cultivating the allochimeric NT unit in the culture to provide allochimeric ES cells. 2. The method according to claim 1, characterized in that the allochimeric ES cells grow in culture under conditions which result in differentiated cells. 3. The method according to claim 2, characterized in that the differentiated cells are neuronal, muscular or hematopoietic cells. 4. The method according to claim 2, characterized in that the species is an ungulate and the female host is an ungulate. 5. The method according to claim 1, characterized in that the human nucleus is from a differentiated human cell. 6. The method according to claim 1, characterized in that the nucleus of a species other than human is introduced by means of electrofusion. The method according to claim 1, characterized in that it comprises the additional step of genetically modifying the allochimeric ES cells. 8. A method for producing allochimeric cells comprising mitochondria of a first human and a nucleus of a second and different human, the method characterized in that it comprises: enucleating an oocyte of a first human; introducing the nucleus of a ungulate into the enucleated oocyte to provide a first chimeric oocyte; expanding the first chimeric cell in the culture to provide at least 4 ES cells; inseminating the ES cell in an ungulate which is at the same time the source of the nucleus and allowing the ES cell to grow to at least one fetus comprising the second chimeric oocytes; harvest and isolate at least one of the second chimeric oocytes; enucleating at least one of the second chimeric oocytes and introducing a human nucleus of a differentiated human cell into the second chimeric oocyte to provide an allochimeric oocyte; and expanding the allochimeric oocyte in the culture to provide allochimeric ES cells. 9. The method according to claim 8, characterized in that the allochimeric ES cells grow in culture under conditions that result in differentiated cells in neuronal, muscular or hematopoietic trajectories. 10. The method according to claim 8, characterized in that it comprises the additional step of genetically modifying the allochimeric ES cells. 11. A method for treating a human host for an indication susceptible to treatment with allochimeric cells, the method characterized in that it comprises: producing differentiated allochimeric cells according to claim 2; and introducing the allochimeric cells into the human host at the site to be treated. 12. The method according to claim 11, characterized in that the allochimeric ES cells are genetically modified before differentiation. The method according to claim 11, characterized in that the human nucleus is from the human host. 14. A composition, characterized in that it comprises a plurality of human allochimeric ES cells. 15. The composition according to claim 14, characterized in that the human alloximeric ES cells are in culture. 16. A composition, characterized in that it comprises a plurality of differentiated allochimeric cells in another different human host. 17. The composition according to claim 16, characterized in that the differentiated allochimeric cells are in culture. 18. The composition according to claim 16, characterized in that the differentiated allochimeric cells are in neuronal, muscular or hematopoietic trajectories. 19. A composition, characterized in that it comprises a mixture of allochimeric ES cells and differentiated allochimeric cells in culture. 20. A composition, characterized in that it comprises a plurality of genetically modified allochimeric ES cells as a result of the in vitro introduction of exogenous DNA into at least one allochimeric ES cell.