CN1673361A - Multipotential cell containing heterogenous nuclear and mitochondrion - Google Patents

Abstract

A heterochimeric ES cell containing a nucleus from one individual of one species and mitochondria from a different individual of the same species is provided. ES cells can be used to differentiate into differentiated cell types for use in therapy, or to study different developmental processes in forming embryos and in differentiation.

Description

Pluripotent cells with heterogeneous nuclei and mitochondria

Field of Invention

The field of the invention is the manipulation of cells to generate non-human oocytes for use in nuclear transfer procedures, wherein the nuclear transfer units contain heterogeneous nuclei and mitochondria from two different cell sources of the same species.

Background of the Invention

It has been demonstrated that the nuclei of differentiated cells can be transplanted into enucleated mature (term) cells, resulting in dedifferentiated pluripotent cells, which offers a wide range of opportunities for biological manipulation, research and therapy. There are increasing opportunities to correct biological deficiencies or adverse medical conditions using cells or organs that can replace or supplement these deficiencies or conditions. As for transplantation, the reliance so far has been on obtaining organs from donors who have died or who have had two organs and have given up one of them. Although people are actively encouraged to provide usable organs after death, there is still an acute shortage of available organs and many people wait in line. In cases where failure to replace a defective organ can be fatal, there is limited time to find a replacement.

Not only are organs in short supply, but donors must be histocompatible with recipients. Even when there is a close histocompatibility match between donor and recipient, the recipient must often rely on immunosuppressive drugs to prevent graft rejection. These drugs often have serious side effects for transplant recipients, but these must be tolerated because the absence of a graft would be fatal. As surgical capabilities have expanded, cell and organ transplants have become more likely to correct a large number of indications.

Embryonic stem ("ES") cells offer the opportunity to produce differentiated cells, organs, and whole individuals on demand, allowing one to clone specific genotypes. For livestock, such as ungulates, nuclei from pre-implantation livestock embryos support the maturation of enucleated oocytes (Smith et al., Biol Reprod. 40:1027-1035 (1989); Keefer et al., supra 50:935 -939(1994)). Due to the significance and importance of ES cells, there are numerous papers reporting different aspects of this technique. For example, Notarianni et al., J. Reprod. Fert. Suppl. 43:255-260 (1991) report the establishment of stable pluripotent cell lines from porcine and sheep blastocysts; Gerfen et al., Anim.Biotech.6: 1-14 (1995) reported the isolation of embryonic cell lines from porcine blastocysts, which differentiated into several different cell types during culture; Cherny et al., Theriogenology 41: 175 (1994) reported long-term culture preservation pluripotent bovine primordial germ cell-derived cell line that forms embryoid bodies and spontaneously differentiates into at least two distinct cell types; reported by Campbell et al., Nature 380:64-68 (1996) in favor of isolating mice Sheep embryos were cultured under the conditions of the ES cell line, and blastoderm (“ED”) cells of the embryos were subjected to nuclear transfer on day 9 to produce live lambs; VanStekelenburg-Hamers et al., Mol.Reprod.Dev.40:444 -454 (1995) reports the isolation and characterization of permanent cell lines from inner cell mass ("ICM") cells of bovine blastocysts; Smith et al., WO 94/24274, published October 7, 1994, Wheeler et al. WO94/26889, published on November 24, 1994, reports the generation of bovine and porcine pluripotent ES cells that can be used to produce transgenic animals; Collas et al., Mol.Reprod.Dev.38:264-267 (1994) reported Nuclear transfer to bovine ICM by microinjection of lysed donor cells into enucleated mature oocytes (see, Keefer et al., supra); Sims et al., Proc. Natl. Acad. Sci. USA 90: 6143-6147 (1993) reported that by transplanting the nuclei of bovine ICM cells cultured for a short period of time in vitro into enucleated mature oocytes, the production of calves (see, Stice et al., Biol.Reprod.54: 100-110 (1996 )). Finally, PCT/US97/12919 by Robl et al. reports ES cells generated by interspecies nuclear transfer.

The exceptional opportunities offered by ES cell manipulation warrant continuous research and improvement of the properties of ES cells, the sources from which they can be derived, the manner in which they proliferate and mature, and so on. Therefore, there is great interest in finding ways to use ES cells broadly in the development of cells and organs or organ fragments for transplantation, animal husbandry, studying developmental processes leading to cell differentiation and formation of organoids, organs and parts thereof.

Invention overview

Provided are methods and compositions relating to modified oocytes and ES cells capable of culturing, proliferating and differentiating into different cell types, engineered ES cells, differentiated cells produced therefrom, organs or parts thereof and organisms produced therefrom . This method involves the use of heterologous oocytes, where heterologous refers to the final species used for cells that are enucleated and introduced into a heterogeneous nucleus, to generate chimeric cells that contain cytoplasm and mitochondria from one species and nuclei from a different species. After chimeric cell proliferation to obtain nuclear transfer (NT) units that can generate ES cells, ES cells can be used for further proliferation and scientific research, or NT units can be transplanted into the uterus of allogeneic, syngeneic, or other compatible heterogeneous hosts (based on primordial oocytes) for pregnancy and delivery. Oocytes from this F1 animal can then be harvested, enucleated, and introduced with heterologous nuclei to produce an oocyte capable of producing single-species protein-containing, immunocompetent cells with reduced risk of rejection upon implantation into an allogeneic host , so it can be accepted. For the purposes of this application, these cells and their progeny are referred to as "allochimeric". Of particular interest is the use of nuclei from hosts to which differentiated heterochimeric cells are to be administered.

Specific implementation plan description

A heterochimeric cell comprising mitochondria from a first individual of a first species and a nucleus from a second individual of the same species is provided. In general, a pluripotent cell capable of developing into an embryo or a number of different cell types will be produced, containing mitochondria from a heterologous species (heterologous species means the species of interest), and a heterogeneous nucleus (heterogeneous means the cell to be implanted species, or a different species compatible with cell implantation and cell differentiation into embryos). Developed or mature embryos can then be used to harvest oocytes, enucleate the oocytes, and introduce nuclei from the same species as mitochondria. The resulting heterochimeric cells can be used to study differentiation in vitro and in vivo for the generation of cells of different differentiation pathways (e.g., ectoderm, endoderm, or mesoderm), and of these classes of cells, such as neuronal cells, neuronal , astrocytes, glial cells, ganglion, etc., hematopoietic cells, such as lymphocytes, macrophages, NK cells, erythrocytes, megakaryocytes, etc., fibroblasts, myoblasts, etc.

The method includes the following main steps:

1. Oocytes are harvested and enucleated.

2. Introduction of nuclear mitochondrial genes from a species different from the egg cell into the nuclear genome of a somatic cell of a different species.

3. Introducing a nucleus from a species other than the egg cell into an enucleated egg cell to produce a chimeric cell.

4. Expansion of chimeric cells to generate competent chimeric ES cells, or incubation to develop nuclear transfer (NT) units.

5. Implantation of active chimeric NT units into compatible female hosts.

6. Growing chimeric NT units to embryos or to term to produce neonatal animals.

7. Make newborn animals grow to a certain age to produce chimeric oocytes.

8. Harvest chimeric oocytes from the offspring.

9. Enucleate chimeric oocytes and introduce nuclei heterologous to oocyte mitochondria to generate heterologous chimeric NT units.

10. Culture heterologous chimeric NT units to generate ES cells.

11. Optionally genetically modifying heterochimeric ES cells.

12. Production of other cell types using heterologous chimeric ES cells.

Each step of the method will now be described in detail.

1. Oocytes are harvested and enucleated.

Egg cells can be harvested from any conventional host of interest, desirably containing mitochondria from the species of interest. Thus, any mammalian species of interest may be used as a source of oocytes, especially domestic animals, more particularly large domestic animals such as horses, cattle, sheep, pigs, cats, dogs, rabbits, mice, etc., and primates, Such as humans, monkeys and apes. Oocytes can be obtained by any conventional method, depending on whether the source of the follicles is slaughtered animals or requires surgical methods. Of particular interest are primate cells, and more particularly human cells, which can be used to generate differentiated cells. These cells can be from any member of these species and the nucleus used subsequently will determine the species and the histocompatibility of the cells. Human cells are particularly useful in the present invention, although there are examples of other species where heterochimeric cells can function, such as exotic animals, animals where in vitro fertilization is not feasible, and the like.

Human cells have been isolated and enucleated. In Zhang et al., J. Assist. Reprod. Genet. 12:361-8 (1995), human fetal eggs were isolated and cryopreserved and found to mature to form polar bodies after thawing. Messinis et al., Br. J. Obstet. Gynaecol. 93: 39-42 (1986); Pellicer et al., Hum. Reprod. 4: 536-40 (1989); Wahlstrom et al., Ann. N. Y. Acad. Sci. 442 : 402-7 (1985); Wood et al., Br. J. Obstet. Gynaecol. 88: 756-60 (1981) describe aspiration of follicles. Enucleation was performed using techniques used for other species in a manner described in the experimental section.

Oocytes can be matured in vitro according to known methods using a suitable maturation medium. Oocytes containing polar bodies (metaphase II oocytes) can be used as host cells. See, eg, Prather et al., Differentiation 48:1-8 (1991) and Seshagine et al., Biol. Reprod. 40:544-606 (1989).

Enucleation is achieved as usual, conveniently using a micropipette to aspirate the polar body and surrounding cytoplasm. Oocytes are then selected for successful enucleation.

2. Introduction of nuclear mitochondrial genes from a species different from the egg cell into the nuclear genome of a somatic cell of a different species.

Somatic nuclei can be constructed by introducing genes critical for mitochondrial function in the type of mitochondria contained in the egg cell. The present invention is particularly useful for introducing human nuclear mitochondrial genes into somatic cells of animals, especially bovines, so as to benefit the cellular functions of animal cells containing human mitochondria.

3. Introducing a nucleus from a species other than the egg cell into an enucleated egg cell to produce a chimeric cell.

Many mammalian hosts can be used as a source of nuclei, a major issue being convenience. In addition to the need for growth and maturation of chimeric NT units, other considerations in selecting a host source are: ease of oocyte acquisition from host, ease of manipulation and growth in culture, maturity of technology, success rate of previous offspring, ease of host growth , the viability of the resulting chimeric oocytes, the number of oocytes available, the compatibility of mitochondria with the host nucleus, and other practical considerations. While any mammalian host may be used, of particular interest are domestic animals, more particularly large domestic animals or non-human primates. Representative mammals include ungulates, cattle and sheep, pigs, cats, horses, rabbits, mice, dogs, and the like. Isolated oocytes were matured in vitro and screened for the presence of polar bodies.

Nuclei can be from any suitable source and can be used to produce germ cells or somatic cells of progeny. Thus, nuclei may be derived from fetal cells, neonatal animal cells, mature cells, cells produced in culture, and the like. Tumor cell nuclei are generally not used, but may be used depending on the nature of the tumor. The source can be differentiated cells, which can be dormant, G ₀ cells, or in an active state, such as G ₁ or G ₂ cells. Cell types include single-celled embryos (zygotes) or pronuclei, egg cells, blastomeres, epithelial cells, endothelial cells, muscle cells, keratinocytes, skin cells, alveolar cells, liver cells, kidney cells, neuronal cells, hematopoietic cells , fibroblasts, endothelial cells, parenchymal cells, adipocytes, glial cells, follicular cells, etc., the choice of which can depend on many factors related to the end use.

Various techniques are available for introducing nuclei into enucleated eggs. Fusion and injection have been shown to be effective, where cells of nuclear origin are placed in the perivitelline space of an enucleated oocyte, fused using electrical pulses or using fine glass needles in a fusion chamber, donor nuclei or from fertilized oocytes The pronuclei of the cells can be isolated and transported into the cytoplasm of the recipient egg. These pronuclei may be from animals whose genomes have been modified to contain nuclear mitochondrial genes of the same species as the recipient oocyte. (See, eg, Prather et al., US Patent No. 4,997,384.) Fusion can also be accomplished using electrical pulses or using different fusogenic agents such as Sendai virus. (See, eg, Graham, Wister Inot. Symp. Monogr. 9:19 (1969); Collas and Barnes, Mol. Reprod. Dev. 38:264-267 (1994)).

4. Expansion of chimeric cells to generate active chimeric ES cells, or incubation to develop nuclear transfer (NT) units.

Following fusion, the resulting fused nuclear transfer ("NT") units are placed in a suitable medium (eg, CRIaa medium) until activated. Activation generally occurs shortly thereafter, usually within 24 hours, more commonly after 4-9 hours. Activation can be accomplished using well-known methods for mammalian cells, such as incubation of NT units at subphysiological temperatures (eg, room temperature), sperm penetration of oocytes, electrical and chemical shock, and the like. See, eg, Susko-Parrish et al., US Patent No. 5,496,720. Other methods include the use of electroshock, ethanol treatment, or chelator treatment to simultaneously or sequentially increase oocyte levels of divalent cations, such as calcium, magnesium, barium, or strontium, as appropriate in the form of ionophores; or to reduce oocyte Phosphorylation of cellular proteins using kinase inhibitors such as 6-dimethylaminopurine (DAMP), staurosporine, roscovitine, butyrolactone, 2-aminopurine, and sphingosine, or to oocytes Introduce one or more phosphatases, such as phosphatase 2A or 2B. An important activation technique is to activate NT units within 18-30 hours of initiation of culture in maturation medium. NT units were then removed from the medium and placed in activation medium (2 mL TL Hepes with 2 mg bovine serum albumin, 5 mM ionomycin and 2 mM 6-DMAP) for 4 minutes on a slide warmer. At the end of 4 min, rinse the NT units with TL Hepes and place in medium containing 2 mM 6-DMAP for 2-5 h at 38.5 °C and 5% _CO2 . At the end, rinse the NT unit 4 times with TL Hepes and place it for culture. When the desired stage of development is reached, ES cells will be generated, or NT units will be transferred into female recipients.

The NT units of activated chimeric cells can then be cultured in an appropriate in vitro or in vivo culture medium until ES cells and cell colonies of developing embryos such as blastocysts are produced. Media suitable for the culture and maturation of ES cells and embryos are well known in the art. Chimeric cells can be treated as cells of a species derived from the nucleus, whose genes determine surface membrane, cytoplasmic, and nuclear proteins. Examples of known media include: Ham's F-10 + 10% Fetal Calf Serum ("FCS"), Tissue Culture Media-199 ("TCM-199") + 10% Fetal Calf Serum, Tyrodesp Albumin-Lactate-Acetone acid ("TALP"), Dulbecco's phosphate buffered solution ("PBS"), Eagle and Whitten medium. A commonly used medium is TCM-199 or DMEM with 5-20% fetal calf serum or factors of similar origin to support growth, such as neonatal serum, estrous cow serum, lamb serum, or steer serum. A representative medium is TCM-199 containing Earl's salts, 10% fetal bovine serum, 0.2 mM sodium pyruvate, and 50 μg/ml gentamicin. These media can be used to provide conditioned media for cell lines using follicular granulosa cells, oviduct cells, BRL cells, uterine cells, and STO cells. In addition, the medium described in Rosenkrans, jr., US Pat. No. 5,096,822 can also be used. This medium is called CR1 and contains about 1-10 mM, usually 1-5 mM hemicalcium L-lactic acid, sodium chloride, potassium chloride, sodium bicarbonate and a small amount of fatty acid-free bovine serum albumin, when added When essential and non-essential amino acids are included, the medium is called CR1aa. A typical CR1 medium contains 114.7 mM NaCl, 3.1 mM KCl, _26.2 mM _Na2CO3 , 5 mM hemicalcium L-lactic acid and 3 mg/ml fatty acid-free bovine serum albumin.

The activated NT unit chimeric ES cells are conveniently placed in CR1aa medium containing 1.9 mM DMAP for about 4 h, subsequently washed with HECM, and then cultured in _CR1aa containing BSA at about 38.5 °C and 5% CO for about 4 h. 4-5 hours. Cultured NT units are typically washed and grown in appropriate conditioned media. An effective medium is CR1aa medium containing 10% FCS and 6 mg/ml BSA. Suitable feeder layers include fibroblasts and epithelial cells, particularly uterine epithelial cells, forming sources such as ungulate, chicken, murine, STO, SI-m220 and BRL cells. Mouse embryonic fibroblasts have been found to be particularly useful. After sufficient time (varies with species of nucleus), chimeric ES cells are obtained and can now be used for implantation into a suitable host.

5. Implantation of active chimeric NT units into compatible female hosts.

6. Growing chimeric NT units to embryos or term to produce neonatal animals.

These stages are well described in the literature for many species, including mice and ungulates. For fertilization, the NT unit is transferred into the uterus. After fertilization, monitor the host to ensure successful implantation of NT units. Depending on the nature of the host, the host can be restrained to prevent spontaneous abortion. Delivery of a fetus or newborn animal may be by natural or induced abortion, natural childbirth or caesarean section.

7. Make newborn animals grow to a certain age to produce chimeric oocytes.

8. Harvest chimeric oocytes from the offspring.

The above stages depend on the nature of the host. The host is cared for normally so that the newborn animal grows into a healthy host. Harvesting of oocytes can be performed as described above.

9. Enucleate chimeric oocytes and introduce nuclei heterologous to oocyte mitochondria to generate heterologous chimeric NT units.

Methods for enucleation and introduction of nuclei have been described. The choice of species to provide the nucleus will depend on the use of the cell. Cells can be used for cloning of specific species, especially species where oocytes are difficult to grow, such as humans or rare species, or as a source of differentiated cells where the cells contain nuclei and mitochondria from the same species. Unlike humans, other species used in the present invention include clones of rare and endangered animals.

10. Culture heterologous chimeric NT units to generate ES cells.

The NT units are cultured on feeder layers until the NT units reach a size suitable for isolating ES cells, usually at least 4 cells, preferably at least 50 cells, and generally not more than 400 cells. The culture generally uses the conditions of 38.5°C and 5% CO ₂ , and the medium is replaced approximately every 1-5 days.

The final stage involves mechanical removal of the NT unit from the culture, isolation of the cells from within the NT unit, although cells from other parts of the NT unit can also be used, washing of the NT unit cells, and seeding the cells on cells from the same or different species as the nucleus. On a feeder layer, such as irradiated fibroblasts. Cells are maintained on feeder layers in appropriate media (eg, α-MEM supplemented with 10% FCS, 0.1 mM β-mercaptoethanol and L-glutamine). Change the growth medium approximately every 1-3 days.

For human heterochimeric ES cells, the individual cells are not well defined and the borders of the colony are refracted and smooth in appearance. Colonies do not have an epithelial-like appearance.

The inner cell mass (ICM) cells of the blastocyst can be generated using NT unit cells. ICM cells can be grown using a feeder layer such as STO (mouse fibroblasts) containing charcoal-adsorbed serum.

11. Optionally genetically modifying heterochimeric ES cells.

ES cells can be modified with DNA according to conventional techniques. Naked DNA, recA-coated DNA, viruses, plasmids, YAC, extrachromosomal DNA, chromosomal fragments, cDNA or other sources of desire The genes, regulatory sequences, etc. are introduced into ES cells. See, eg, Schneike et al., Science 278:2130-3 (1997).

Modifications that provide constitutive or inducible expression of specific products such as insulin, angiogenic factors, cytokines such as interleukins, interferons and colony stimulating factors, blood factors such as serum albumin, thrombin, fibrinogen, thrombopoiesis hormone, erythropoietin, tissue plasminogen activator, etc., hormones, such as growth hormone, growth factors, such as epidermal growth factor, basic fibroblast growth factor, glial cell-derived neurotrophic growth factor, etc., enzymes , enzyme inhibitors, such as α-antitrypsin, telomere-associated proteins, such as Sir, telomerase, etc., neuronal proteins, such as neurotrophic factor-3, 4/5, ciliary neurotrophic factor, etc., trypsin, Basic proteins, taste bud proteins, eye proteins, hemoglobin, transcription factors, lipoproteins, immunoglobulins, surface membrane receptors such as insulin receptors, oncogene inhibitors, L-dopamine and angiogenesis inhibitors. Furthermore, homologous recombination of the nucleus can be used to generate heterochimeric ES cells in order to correct defective or undesired phenotypes. For example, nuclei from patients with sickle cell anemia can be genetically modified to obtain cells that produce native hemoglobin; patients with hemophilia can correct mutated genes for blood factors (such as factor VIIIc or VIIIvw, Chrismas factor); due to genetic factors Patients predisposed to disease may modify the nucleus to produce alleles that are less susceptible to these diseases (eg, AIDS, juvenile onset diabetes, muscular dystrophy, Alzheimer's disease, Parkinson's disease, etc.).

Sometimes instead of importing a heredity, it may be desirable to turn off the heredity or make the gene inducible. Illustrative genes that can be knocked out include oncogenes, histocompatibility proteins, blood group proteins, and other genes whose dominant expression leads to pathology. Homologous recombination can be used or cells can be selected for which the desired knockout has occurred.

12. Production of other cell types using heterologous chimeric ES cells.

Pedersen, Reprod. Fertil. Dev. 6:543-52 (1994) reviewed the extensive literature on ES cell differentiation. The authors report the generation of differentiated cells expressing cell-type-specific CD markers, and the stage of differentiation or cell products that cells do not normally produce at the level of ES cell differentiation. The development of ES cells to specific cell types can be seen from Bain et al., Dev.Biol.168:342-357 (1995) (nerve cells); Palacios et al., Proc.Natl.Acad.Sci.USA 92:7530-7537 (1995 ) (hematopoietic cells); Rathjen et al., Reprod. Fertil. Dev. 10:31-47 (1998).

Target cells 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 related to sensation, such as Taste buds, inner ear cells, bone cells, kidney cells, liver cells, pancreatic cells such as beta-islet cells, fibroblasts and chondrocytes.

Differentiated ES cells can be used in a variety of ways. Cells containing heterologous mitochondria and nuclei provide normal cells containing DNA from a single species. This avoids the problem of incompatibility between the mitochondria and the nucleus, the proteins present in the cytoplasm are all from the same species, the interaction between the nucleus and the mitochondria, such as the transport of proteins is natural, and the immunodominant sequences present on the cell surface are all derived of the same species. Furthermore, cellular processes that depend on the interaction of mitochondria with other intracellular compartments are also natural.

ES cells and differentiated cells offer many opportunities to analyze cellular processes, responses to exogenous factors (e.g. genes, drugs, factors, etc.), to screen for the role of these exogenous factors in drug development, to identify and respond to these exogenous factors Responses associated with specific alleles or mutations, study transcriptional and expression changes caused by different factors.

In addition, because ES cells can be maintained in culture and can proliferate, differentiated cells can be reproducibly produced, which will be based on the same genotype. The present invention also provides the ability to use nuclei from individuals for research and diagnosis. Thus, the susceptibility of a particular individual to external factors such as carcinogens, allergens, toxins, and mutagens can be determined where an intact cell of a species has the native metabolism of that species. Cellular responses to specific regimens, such as drug regimens, can also be studied to determine the effect on normal cells in a particular patient. Regarding the chronic application of the protocol, there is sufficient time to introduce nuclei from differentiated cells from a specific individual of the species using stored enucleated eggs of the species of interest as containers. The ES cells can then be used to generate different differentiated cells at different stages of differentiation that can be subjected to the protocol and alterations as cells tested.

The invention can be used to study mitochondrial and specific nuclear processes. For example, when individuals may contain nuclei deficient in producing proteins used by mitochondria, the method can be used to clone these cells and observe the effects of differentiation on mitochondrial processes and on growth patterns, phenotypes, etc.

In cases where ES cells or NT units are used for embryo maturation and newborn animal production, the use of native mitochondria avoids incompatibility of the resulting individuals when mated with other individuals of the same species. Thus, the fetus is less likely to be rejected by the mother and less likely to have an immune response to the host cell due to the presence of exogenous mitochondrial proteins.

A composition useful in the present invention is an isolated and cultured oocyte containing mitochondria from one species and a nucleus from a different species. Most of the time, nuclei and mitochondria are from fundamentally different species, often from different genera, or even different families. Nuclei are conveniently selected to provide useful sources of enucleated ova, such as domestic animals (ungulates), such as cattle, sheep, and pigs, and experimental animals, such as mice. The present invention also includes eggs containing mitochondria from one individual and nuclei from a different individual of the same species, ES cells produced therefrom, cultures containing these ES cells, differentiated cells produced therefrom, and eggs containing these differentiated cells Cultures.

These compositions contain heterogeneous chimeric oocytes and ES cells in growth culture for cell proliferation. These compositions also comprise admixtures of heterochimeric cells and inner cell mass differentiated cells in a medium for cell differentiation, or concomitant growth or maintenance of ES cells and differentiated cells.

For hematopoietic cells, in Costar six-well culture plates, a medium containing mitomycin C-treated (5-10 μg/ml, 37°C, 3-4 hours) or irradiated (2-4×10 ³ La De γ-ray; 1 rad = 0.01Gy) RP.0.10 monolayer of bone marrow stromal cells containing recombinant interleukin-3 (rIL-3) (100-300 units/ml), rIL-6 and F (10% final concentration v/v), supernatant of a 2-day culture of confluent FLS4.1 fetal liver stromal cells. FLS4.1 supernatant contains FLT3 ligand, steel factor and a new factor that supports the growth of hematopoietic stem cells, in 2-2.5ml medium [Iscove's Dulbecco's modified medium/50μM 2-mercaptoethanol/2mM L-glutamine /50 μg/ml gentamicin/7.5% v/v FCS], 37°C, 7.5% CO ₂ /92.5% air. Cells were collected every 5-7 days and subcultured in new Costar six-well culture plates containing mitomycin C-treated RP.0.10 stromal cells and freshly prepared cytokine-containing medium. (Palacios et al., Proc. Natl. Acad. Sci. USA 92:7530 (1995)). It should be understood that other cell lines that provide similar conditioned media, and other components that provide similar activities, may also be used.

For neuronal cells, ES cells were subjected to an 8-day induction procedure consisting of 4 days of culture into retinoic acid (RA)-free aggregates, followed by 4 days of culture in the presence of RA. The medium was DMEM (high glucose with L-glutamine, without pyruvate; GIBCO11965-043), 10% fetal calf serum, 10% neonatal calf serum and nucleoside stock solution.

Other media can also be used to direct differentiation to other cell types, using factors that occur naturally and affect differentiation in vivo, such as hematopoietic factors, such as G-CSF, M-CSF, GM-CSF, interleukins, interferons, and other cytokines and growth factor.

Enucleated oocytes, all containing identical mitochondria, can be frozen in appropriate media for extended periods of time and carefully thawed before use.

These differentiated cells, whether genetically modified or not, can be used therapeutically, and healthy cells can be directed into the appropriate compartment to perform the desired function. For example, myocytes may be used for transplantation into cardiac muscle tissue or other muscle tissue, while native or genetically modified cells may have the advantage of being able to correct mutant proteins, eg in muscular dystrophy. See, eg, US Patent No. 5,602,301. Hematopoietic cells, such as lymphocytes, natural killer cells, megakaryocytes, eosinophils, basophils, monocytes, or their precursors, which can be specific cell types or can be multiple, can be infused into the patient capable of, and will differentiate into, a large number of different types of cells. Beta-islet cells can be transplanted onto the pancreas, or placed in protective containers where they can be innervated by the blood supply, for the treatment of diabetes. Neuron cells can be introduced into the brain cavity to treat Parkinson's disease, Alzheimer's disease, cerebral palsy, etc. by providing sources of L-dopamine, NGE or other factors or other compositions that can affect brain function. Other indications for which ES cells or their differentiated progeny are suitable include: spinal cord injury, multiple sclerosis, liver disease, vascular disease, cartilage replacement in burns, heart disease, kidney disease, urinary tract disease, prostate disease, and diseases caused by aging.

These cells can be used for enhanced constitutive or inducible supply of specific factors. Pruschy et al., Chem. Biol. 1: 163-72 (1997) improved by inducing production of the product by oral administration of a pill of activatable transcription factors. Thus, tissue plasminogen activator can be induced for heart attack, insulin for diabetes, and lymphocyte activation for infection by introducing non-human receptors such as ecdysone receptors.

The following examples are for illustration and not limitation.

experiment

Materials and Methods

recipient human oocyte

Oocytes from Human Fetus: Fetal ovaries were obtained from fetuses at 16-20 weeks of gestation after elective abortion. Ovarian tissues were minced into ~1 mm size and cultured in Waymouth medium supplemented with 15% (v/v) fetal bovine serum, 0.03 IU/ml FSH and 35 ng/ml insulin. Tissues were cultured at 37°C and 5% _CO2 air for 5-25 days in Falcon dishes and 3-40 days on Costar Transwell-COL membranes, after which final maturation was induced in the presence of LH and human follicular fluid. Monolayer fragments composed of fibroblasts form fresh tissue within 2-3 days of culture. After 1 week in culture, follicles separated from the ovarian tissue but remained attached to the monolayer. The maximum number of follicles separated from the tissue appeared about 1 week after the initiation of culture. After 40 days of culture in the Costar dish, the diameter of the main part of the egg reached more than 80 μ, and about one-third was surrounded by the zona pellucida. Extrusion of the first polar body was observed in eggs grown in Costar plates for 40 days after induction of final maturation. Mature eggs can be collected and directly enucleated.

Oocytes from mature females. Human eggs were obtained using the technique of Trotnow et al., Arch. Gynecol. 236:211-7 (1985). Using the suction needle passing through the trocar, the suction needle is manipulated by the converter of the DiasonicsDS 1 sector scanner, and continuous ultrasound imaging is performed on the suction needle. The patient is given an epidural anesthesia.

Other techniques can also be used, including administration of follicle stimulating hormone according to the protocol described by Mercan et al., Hum. Reprod. 12:1886-9 (1997).

Oocytes can then be matured in vitro as follows. The immature oocytes were washed with TL-HEPES buffered medium containing 3 mg/ml bovine serum albumin (fraction V). Oocyte mound complexes were placed in TCM-199 containing 10% fetal calf serum, LH and/or FSH and estradiol at 39°C. After approximately 20 hours in maturation medium, oocytes were removed, placed in TL-HEPES containing 1 mg/ml hyaluronidase, and cumulus cells were removed by repeated pipetting through a fine-bore pipette. The stripped oocytes were screened according to the polar bodies, and oocytes containing polar bodies (metaphase II oocytes) were selected for further application.

Bovine donor cells:

Heifers are superovulated and artificially inseminated. Embryos were harvested from the reproductive tract on day 5 or 6 of estrus. Embryos were rinsed from the reproductive tract with PBS and recovered. Embryos matured in vitro, fertilized and cultured were used as donor embryos on day 5 after fertilization. The insemination procedure was described by Keefer et al., Mol. Reprod. Dev. 36:469-74 (1993).

Enucleated:

Enucleation was performed approximately 18 hours after the onset of maturation (hpm) using an angled micropipette. Enucleation was confirmed in TL-HEPES medium plus bisphenolthiamine (Hoechst 33342, 3 μg/ml).

Nuclear transfer:

Individual donor cells are placed in the perivitelline space of the recipient enucleated oocyte. Human oocyte cytoplasm and bovine donor nuclei (NT units) were fused together using the electrofusion technique: 90V fusion pulses for 25 microseconds 24 hours after the onset of oocyte maturation. The resulting NT units were placed in CR1aa medium until 28 hours after initiation of maturation, at which point they were activated as follows. NT units were exposed to ionomycin-6-DMAP (2 mM) for 4 min, followed by DMAP (2 mM) alone for 4 h in TL-HEPES supplemented with 1 mg/ml serum albumin, followed by supplementation with 30 mg/ml serum Albumin was washed with TL-HEPES for 5 minutes. The NT units were then transferred to a small drop of CR1aa medium plus 10% FCS and 6 mg/ml serum albumin on a 4-well plate containing a confluent feeder layer of mouse embryonic fibroblasts. NT units were cultured at 38.5 °C and 5% _CO2 for more than 3 days. Medium was changed every 3 days until day 12 after the initiation of activation. At this point approximately 50 cells should have formed which are mechanically removed from the zona pellucida and used to generate embryonic cell lines.

Mouse embryonic fibroblast feeder layer:

Primary cultures of mouse embryonic fibroblasts were obtained from 14-16 day old mouse fetuses. After aseptically removing the head, liver, heart, and digestive tract, the embryos were minced and incubated at 37°C in a pre-warmed trypsin-EDTA solution (0.05% trypsin/0.02% EDTA; GIBCO, Grand Island, NY) 30 minutes. Fibroblasts were seeded in tissue culture flasks and cultured in α-MEM medium (Bio Whittaker, Walkersville, MD) supplemented with 10% FCS, penicillin (100 IU/ml) and streptomycin (50 μl/ml). 3-4 days after passage, embryonic fibroblasts in 35 x 10 Nunc dishes (Baxter Scientific, McGaw Park, IL) were irradiated. Irradiated fibroblasts were grown and stored at 37 °C in a humidified atmosphere containing 5% _CO2 . Plates containing a homogeneous cell monolayer are used to grow embryonic cell lines.

Generation of embryonic cell lines:

NT unit cells obtained as described above were washed and plated directly on irradiated feeder fibroblasts (see above). These cells include the inner cells of the NT unit. Cells were maintained in α-MEM growth medium supplemented with 10% FCS and 0.1 mM β-mercaptoethanol. Change the medium every 2-3 days. The first colonies were observed by day 2 or day 3 of culture. The colonies proliferated and displayed similar morphology to previously published mouse and bovine embryonic stem (ES) cells. Individual cells within the colony are not well defined, and the borders of the colony are refracted and smooth in appearance. These cells have an epithelial appearance.

Generation of Chimeric Bovine NT Units:

Chimeric NT units are selected and implanted into cows as follows: 1-5 NT units are implanted in the uterus. The resulting calf grows to the appropriate stage of oocyte development, at which point the eggs are aspirated and isolated. Alternatively, oocytes can be obtained from adult females or from slaughtered cow ovaries by superovulation and ultrasound-guided oocyte retrieval.

Generation of heterologous chimeric human cells:

Chimeric eggs obtained as described above are processed as described above for growth and enucleation, and are then prepared to receive human nuclei. Human epithelial cells were gently scraped from the oral cavity of consenting adults using standard glass slides. Rinse the cells from the slide into a Petri dish containing PBS with Ca or Mg. Aspirate cells through a small-bore pipette to break up cell clumps into a single-cell suspension. Cells were then transferred to a small drop of TL-HEPES medium containing 10% FCS under oil, and nuclear transfer was performed into enucleated chimeric bovine oocytes containing human mitochondria using the method described above.

Heterochimeric cells can use nuclei from essentially any suitable human cell source, such as fibroblasts, epithelial cells, keratinocytes, blood lymphocytes, or bladder epithelial cells. Heterochimeric cells were grown in culture, according to different modes of differentiation, as described by Stice et al., (1998), supra, including the preparation of myocytes.

According to the present invention, there is provided a heterologous chimeric human cell comprising human nuclei and human mitochondria from different individuals. These cells are versatile because they provide a mimic of natural human cells for the study of cellular processes in embryonic development and differentiation to differentiated cell types. These cells can also be used to produce differentiated cells for cell therapy or production of human factors. These cells are cells that only produce human proteins so as to be unlikely to induce an immune response, allow genetic manipulation of the nucleus, provide human cells that can be introduced into an individual as the source of the nucleus, but have the nucleus modified for therapeutic or other use, and provide genetic A source of genotype-identical cells that can be used for cloning of specific genotypes.

References cited in this application are hereby incorporated by reference. All procedures and methods, including those described, constitute the method part of this application, and are applicable to the subject matter of this application according to those skilled in the art.

Now that the invention has been fully described, it will be apparent to those skilled in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims (20)

1. method of producing the allos chimeric cell, it contains from first people's plastosome and nuclear from second different people, and this method comprises:

Will be from first people's ovocyte stoning;

To import from the nuclear of inhuman species in this enucleation oocyte, produce first kind of chimeric ovocyte;

Cultivate first kind of chimeric cell of amplification, produce NT unit;

This ES cell is implanted in the compatible female host, made this NT unit be grown to the embryo that at least one contains second kind of chimeric ovocyte;

Collection also separates at least one second kind of chimeric ovocyte;

With at least one second kind chimeric ovocyte stoning, and people's nuclear imported in this second kind of chimeric ovocyte, produce the chimeric NT unit of allos; With

Cultivate the chimeric NT unit of this allos, produce the chimeric ES cell of allos.

2. according to the process of claim 1 wherein that the chimeric ES cell of this allos is causing producing incubation growth under the condition of noble cells.

3. according to the method for claim 2, wherein this noble cells is neuronal cell, myocyte or hematopoietic cell.

4. according to the method for claim 2, wherein these species are ungulates, and this female host is a ungulate.

5. according to the process of claim 1 wherein that people's nuclear derives from the human cell of differentiation.

6. merge the nuclear that imports from inhuman species according to the process of claim 1 wherein by electricity.

7. according to the method for claim 1, comprise the step of the chimeric ES cell of this allos of genetic modification in addition.

8. method of producing the allos chimeric cell, it contains from first people's plastosome and nuclear from second different people, and this method comprises:

Will be from first people's ovocyte stoning;

To import from the nuclear of ungulate in this enucleation oocyte, produce first kind of chimeric ovocyte;

Cultivate first kind of chimeric cell of amplification, produce at least 4 ES cells;

This ES cell is implanted in the ungulate identical with the nuclear source, made this ES cell be grown to the embryo that at least one contains second kind of chimeric ovocyte;

Collection also separates at least one second kind of chimeric ovocyte;

With at least one second kind chimeric ovocyte stoning, and will import from the nuclear of differentiation of human class cell in this second kind of chimeric ovocyte, produce the chimeric ovocyte of allos; With

Cultivate the chimeric ovocyte of this allos of amplification, produce the chimeric ES cell of allos.

9. method according to Claim 8, wherein incubation growth under the condition of the noble cells of the chimeric ES cell of this allos in producing neurone, muscle or hematopoiesis approach.

10. method according to Claim 8 comprises the step of the chimeric ES cell of this allos of genetic modification in addition.

11. treat the method for in the human host allos chimeric cell being treated responsive indication for one kind, this method comprises:

Generation is according to the allos chimeric cell of the differentiation of claim 2; With

With a position of this allos chimeric cell importing human host, treat this indication.

12. according to the method for claim 11, wherein the chimeric ES cell of this allos is genetically modified before differentiation.

13. according to the method for claim 11, wherein people's nuclear derives from this human host.

14. composition that contains the chimeric human ES cell of a large amount of allos.

15., wherein cultivate the chimeric human ES cell of this allos according to the composition of claim 14.

16. a composition, it contains in a large number the allos chimeric cell from non-human host's differentiation.

17., wherein cultivate the allos chimeric cell of this differentiation according to the composition of claim 16.

18. according to the composition of claim 16, wherein the allos chimeric cell of this differentiation is in neurone, muscle or the hematopoiesis approach.

19. a composition, it contains the chimeric ES cell of allos of cultivation and the mixture of the chimeric noble cells of allos.

20. a composition, owing to external importing foreign DNA in the chimeric ES cell of at least one allos, it contains the chimeric ES cell of allos of a large amount of genetic modifications.