CN1673361A - Multipotential cell containing heterogenous nuclear and mitochondrion - Google Patents
Multipotential cell containing heterogenous nuclear and mitochondrion Download PDFInfo
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- CN1673361A CN1673361A CNA2005100527010A CN200510052701A CN1673361A CN 1673361 A CN1673361 A CN 1673361A CN A2005100527010 A CNA2005100527010 A CN A2005100527010A CN 200510052701 A CN200510052701 A CN 200510052701A CN 1673361 A CN1673361 A CN 1673361A
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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
Invention field
The field of the invention is the operation of pair cell, is used for the non-human ovocyte of nuclear transplantation method with generation, and wherein allos nuclear and the plastosome from two different cells sources mutually of the same race contained in these nuclear transplantation units.
Background of invention
Confirmed and can produce the pluripotent cell that dedifferentes with the nuclear transplantation of noble cells in stoning maturation (term) cell, this provides chance widely for biologic operation, research and treatment.Use and to substitute or the cell of additional biological defective or unfavorable medical conditions or the chance that organ is corrected these defectives or illness get more and more.As for transplanting, still depend on the organ that obtains from donor so far, wherein donor is dead, and perhaps donor has two parts of organs and abandons a copy of it.Although actively encourage people that operable organ is provided after death, utilizable organ is extremely shortage still, and many people wait in line.Replacing under the fatal situation of defective organ failure possibility, find the time of substitute organ very limited.
Not only organ short supply, and donor must be compatible with receptor tissue.Even between donor and acceptor, have when histocompatibility is mated closely, acceptor also must rely on immunosuppressive drug usually and prevent transplant rejection.These medicines have severe side effect usually to transplant recipient, but must stand, and will be fatal because there is not graft.Because the operation ability expands, it is increasing that cell and organ graft are corrected the possibility of a large amount of indications.
The embryo does (" ES ") cell the chance of producing noble cells, organ and complete individuality as required is provided, and makes people can clone the specific gene type.As for domestic animal, ungulate is for example supported enucleation oocyte reach maturity (people such as Smith, Biol Reprod.40:1027-1035 (1989) from the nuclear of livestock embryo before the implantation; People such as Keefer, the same 50:935-939 (1994)).Because the significance and importance of ES cell has a large amount of papers to report the different aspect of this technology.For example, people such as Notarianni, J.Reprod.Fert.Suppl.43:255-260 (1991) have reported the foundation from the stable pluripotent cell system of pig and sheep blastocyst; People such as Gerfen, Anim.Biotech.6:1-14 (1995) have reported from the pig blastocyst and have separated embryo cell line that these cells are divided into several different cellular types in culturing process; People such as Cherny, Theriogenology 41:175 (1994) have reported the multipotency ox archeocyte deutero-clone that long-term cultivation is preserved, and their form embryoid and spontaneously are divided at least two kinds of different cellular types; People such as Campbell, Nature 380:64-68 (1996) have reported under the condition that helps separating the ES clone in the mouse and have cultivated sheep embryo, carry out nuclear transplantation at the 9th day blastodisc to the embryo (" ED ") cell, produce the lamb that lives; People such as VanStekelenburg-Hamers, Mol.Reprod.Dev.40:444-454 (1995) have reported and have separated inner cell mass (" the ICM ") cell from the ox blastocyst and characterize permanent cell line; People such as Smith, WO announced on October 7th, 94/24274,1994, people such as Wheeler, WO94/26889 announced, and had reported to can be used for producing the ox of transgenic animal and the generation of pig multipotency ES cell on November 24th, 1994; People such as Collas, Mol.Reprod.Dev.38:264-267 (1994) reported by microinjection cracked donorcells in the stoning mature oocyte, and ox ICM is carried out nuclear transplantation (referring to, people such as Keefer, with above); People such as Sims, Proc.Natl.Acad.Sci.USA 90:6143-6147 (1993) reported by with the nuclear transplantation of the ox ICM cell of external Short-term Culture in the stoning mature oocyte, the production calf (referring to, people such as Stice, Biol.Reprod.54:100-110 (1996)).At last, people's such as Robl PCT/US97/12919 has reported the ES cell that is produced by inter-species nuclear transplantation.
The special chance that the ES cell manipulation provides guaranteed to the ES cell characteristic, they can from the lasting research and the improvement of source, their propagation and sophisticated mode etc.Therefore, very pay close attention to and seek the method that in cell and organ or organ fragment are grown, is extensive use of the ES cell, the growth course that be used to transplant, animal rearing, research causes cytodifferentiation and organoid, organ and part thereof to form.
Summary of the invention
Following method and composition is provided, and they relate to can cultivate propagation and be divided into the modification ovocyte and the ES cell of different cellular types, through engineering approaches ES cell, by the noble cells of its generation, organ or its part and by the biology of generation.This method comprises uses the allos ovum, and wherein allos is meant that final species (these cell stonings are also the gone into xenogenesis nuclear) generation that is used for cell contains from the tenuigenin of a kind and plastosome with from the chimeric cell of nuclear not of the same race.After chimeric cell propagation obtains to produce nuclear transplantation (NT) unit of ES cell, the ES cell can be used for further propagation and scientific research, perhaps NT unit can be transplanted in allos, homology or other the compatible xenogenesis host uterus (based on original ovocyte) in order to gestation and childbirth.Can gather in the crops the ovocyte from this F1 animal then, stoning imports allos nuclear, produce a kind of ovum, this cell can produce the protein that contains single species, immunocompetent cell, repels dangerous the reduction when implanting the allosome host, thereby can accept.For the application, these cells and offspring thereof are called as " allos is chimeric " (allochimeric).Special concern will be used the purposes of the host's who breaks up the allos chimeric cell nuclear.
Specific embodiments is described
Following allos chimeric cell is provided, and it contains from first individual plastosome of first species with from the nuclear of second individuality of same species.In general, the pluripotent cell that can develop into embryo or a large amount of different cellular types will be produced, it contains from the plastosome of allos species (the allos species are meant the purpose kind), with xenogeneic nuclear (xenogenesis is meant the species that will implant cell, or implants with cell and cytodifferentiation is the compatible different plant species of embryo).Grow or sophisticated embryo can be used for gathering in the crops ovocyte then, make the ovocyte stoning, and import nuclear from same species as plastosome.The allos chimeric cell that produces can be used for the research differentiation in vitro and in vivo, be used to produce the cell of different differentiation pathway (for example ectoderm, entoderm or mesoderm), cell with these classifications, as neuronal cell, neurone, stellate cell, neurogliocyte, neuroganglion etc., hematopoietic cell, for example lymphocyte, scavenger cell, NK cell, red corpuscle, megalokaryocyte etc., inoblast, sarcoplast etc.
This method comprises following key step:
1. results ovum and stoning.
2. will import in the somatic nuclear gene group of different plant species from the nuclear gene encoding mitochondrial of the species that are different from this ovum.
3. will import in the enucleated oocyte from the nuclear of the species that are different from this ovum, produce chimeric cell.
4. the amplification chimeric cell produces active (competent) chimeric ES cell, and perhaps incubation is to grow nuclear transplantation (NT) unit.
5. the chimeric NT unit of activity is implanted in the compatible female host.
6. make chimeric NT unit be grown to embryo or extremely mature, produce new born animal.
7. make animal neonatal growth to a dating, produce chimeric ovocyte.
8. from the offspring, gather in the crops chimeric ovocyte.
9. with chimeric ovocyte stoning, import and the allogenic nuclear of ovocyte plastosome, produce the chimeric NT unit of allos.
10. cultivate the chimeric NT unit of allos and produce the ES cell.
11. the chimeric ES cell of genetic modification allos randomly.
12. utilize chimeric other cellular type of ES cells produce of allos.
Each step of this method will be described in detail in detail now.
1. results ovum and stoning.
Ovum can be gathered in the crops from any conventional purpose host, wishes to contain the plastosome from the purpose species.Therefore, any purpose Mammals kind all can be used as the source, particularly domestic animal of ovum, heavy livestock more particularly, for example horse, ox, sheep, pig, cat, dog, rabbit, mouse etc., and primate, for example people, monkey and ape.Ovocyte can obtain with any ordinary method, depends on that the source of ovarian follicle is slaughtered animal or needed operation method.Special concern be primates zooblast, human cell more particularly, they can be used for producing the cell of differentiation.These cells can be from arbitrary member of these species, and the nuclear of Shi Yonging will determine the histocompatibility of species and cell subsequently.The present invention especially uses the human cell, although the example of active other species of allos chimeric cell, for example rare animal, infeasible animal in vitro fertilization etc. are also arranged.
The human cell has separated and stoning.People such as Zhang, among the J.Assist.Reprod.Genet.12:361-8 (1995), separate and frozen human tire ovum, melting the ripe polar body that forms of back discovery energy.People such as Messinis, Br.J.Obstet.Gynaecol.93:39-42 (1986); People such as Pellicer, Hum.Reprod.4:536-40 (1989); People such as Wahlstrom, Ann.N.Y.Acad.Sci.442:402-7 (1985); People such as Wood, Br.J.Obstet.Gynaecol.88:756-60 (1981) has described the sucking-off of ovarian follicle.The stoning technology is used for the technology of other species, and concrete mode is described at experimental section.
Ovum can use suitable maturation medium at maturation in vitro in accordance with known methods.The ovocyte (II ovocyte in mid-term) that contains polar body can be used as host cell.Referring to, for example, people such as Prather, people such as Differentiation 48:1-8 (1991) and Seshagine, Biol.Reprod.40:544-606 (1989).
Stoning realizes according to ordinary method, uses micropipet sucking-off polar body and tenuigenin on every side easily.Select ovocyte to finish the stoning of success then.
2. will import in the somatic nuclear gene group of different plant species from the nuclear gene encoding mitochondrial of the species that are different from this ovum.
Can the construct nucleus by importing for the vital gene of mitochondrial function in the contained plastosome type of ovum.The present invention is particularly useful for particularly importing human nuclear gene encoding mitochondrial in the somatocyte of ox to animal, is beneficial to contain the cell function of the zooblast of human mitochondrial.
3. will import in the enucleated oocyte from the nuclear of the species that are different from this ovum, produce chimeric cell.
Many mammalian hosts can be as the source of nuclear, and a subject matter is convenient.Except the necessity of chimeric NT unit growth and maturity, select other consideration in host source to have: to be easy to obtain ovocyte from the host, easy handling and incubation growth, the maturity of technology obtained offspring's success ratio in the past, was easy to host's growth, the viability of the chimeric ovocyte that produces, available ovocyte quantity, the consistency of plastosome and host's nuclear, and other actual consideration.Although can use any mammalian hosts, special concern be domestic animal, more particularly heavy livestock or inhuman primate.Representative Mammals comprises ungulate, ox and sheep, pig, cat, horse, rabbit, mouse, dog etc.Isolating ovocyte is at maturation in vitro, according to the existence screening of polar body.
Endorse with from any suitable source, can be used to produce offspring's sexual cell or somatocyte.Therefore, endorse with from cell that produces in fetus cells, new born animal cell, mature cell, the substratum etc.Generally do not use neoplastic cell nuclei, but can use yet according to the character of tumour.The source can be the cell of differentiation, can be dormancy, G
0Cell perhaps is in active condition, as G
1Or G
2Cell.Cellular type comprises unicellular embryo (zygote) or pronucleus, ovum, blastomere, epithelial cell, endotheliocyte, myocyte, keratinocyte, skin cells, alveolar cell, liver cell, nephrocyte, neuronal cell, hematopoietic cell, inoblast, endotheliocyte, parenchyma, adipocyte, neurogliocyte, follicular cell etc., and their selection can be depending on the many factors relevant with end-use.
There are multiple technologies to can be used in enucleated oocyte, importing nuclear.Fusion and injection have shown it is effective, wherein place the ovum week crack of enucleation oocyte as the cell in nuclear source, utilize electricimpulse or use thin glass needle in merging the chamber, to merge, donor nuclei or separated from the preceding nuclear energy of fertilized oocyte, and be transported in the tenuigenin of acceptor ovum.The animal of the nuclear gene encoding mitochondrial of the species identical with the acceptor ovocyte can modified thereby comprised to these pronucleus from genome.(referring to, for example, people such as Prather, U.S. Patent number 4,997,384.) merge and also can utilize electricimpulse or use different fusogens (as Sendai virus) to finish.(referring to, for example, Graham, Wister Inot.Symp.Monogr.9:19 (1969); Collas and Barnes, Mol.Reprod.Dev.38:264-267 (1994)).
4. the amplification chimeric cell produces active chimeric ES cell, and perhaps incubation is to grow nuclear transplantation (NT) unit.
After the fusion, the fusion nucleus that produces is transplanted (" NT ") unit place suitable medium (as the CRIaa substratum) up to activation.Generally promptly activating in the near future, in 24 hours, more commonly is after 4-9 hour usually.Activation can utilize the known method for mammalian cell to finish, and as cultivating NT unit down at inferior physiological temp (as room temperature), sperm penetrates ovocyte, electricity and chemical shock etc.Referring to, for example, people such as Susko-Parrish, U.S. Patent number 5,496,720.Other method comprises utilizes electroshock, Ethanol Treatment or sequestrant to handle, simultaneously or improve the level of divalent cation in the ovocyte continuously, for example calcium, magnesium, barium or strontium, suitable to ionophoric form; Or the phosphorylation of cell protein in the minimizing ovocyte, use kinase inhibitor, for example 6-dimethylaminopurine (DAMP), Staurosporine, roscovitine, butyrolactone, 2-aminopurine and sphingosine, perhaps in ovocyte, import one or more Phosphoric acid esterases, as Phosphoric acid esterase 2A or 2B.A kind of important activation technique is: activate NT unit after beginning to cultivate in maturation medium in 18-30 hour.From substratum, take out NT unit then, place and activate in the substratum (2mL TL Hepes contains 2mg bovine serum albumin, 5mM ionomycin and 2mM 6-DMAP), in slide glass warmer last 4 minute.4 minutes when finishing, with TL Hepes rinsing NT unit, at 38.5 ℃ and 5%CO
2Placed down substratum 2-5 hour that contains 2mM 6-DMAP.During end, use TL Hepes rinsing NT unit 4 times, place and cultivate.When reaching the etap of hope, will produce the ES cell, perhaps NT unit is transferred in the female receptor.
Can in suitable external or culturing in vivo base, cultivate the NT unit of activated chimeric cell then, up to the cell colony that produces ES cell and embryonic development (as blastocyst).Be suitable for ES cell and embryo culture and sophisticated substratum is well-known in this area.Chimeric cell can be used as from the cell of the species of nuclear and handles, and nuclear gene is determined surface membrane protein, tenuigenin and nucleoprotein.The example of known substratum comprises: Ham ' s F-10+10% foetal calf serum (" FCS "), tissue culture medium (TCM)-199 (" TCM-199 ")+10% foetal calf serum, Tyrodesp albumin-lactic acid-pyruvic acid (" TALP "), Dulbecco phosphoric acid buffer (" PBS "), Eagle and Whitten substratum.A kind of substratum commonly used is TCM-199 or DMEM and 5-20% foetal calf serum or the factor of supporting the similar source of growth, as newborn serum, oestrus cow serum, lamb serum or steer serum.A kind of representative substratum is the TCM-199 that contains Earl salt, 10% foetal calf serum, 0.2mM Sodium.alpha.-ketopropionate and 50 μ g/ml gentamicins.These substratum can be used for clone provides conditioned medium, uses follicular cell, oviduct cell, BRL cell, uterine cell and STO cell.In addition, also can use Rosenkrans, jr., U.S. Patent number 5,096,822 described substratum.This substratum is called as CR1, half calcium (hemicalcium) the L-lactic acid, sodium-chlor, Repone K, sodium bicarbonate and a small amount of FAF bovine serum albumin that contain the 1-10mM that has an appointment, common 1-5mM, when adding essential and non-essential amino acid, substratum is called as CR1aa.A kind of typical C R1 substratum contains 114.7mM NaCl, 3.1mM KCl, 26.2mM Na
2CO
3, 5mM half calcium L-lactic acid and 3mg/ml FAF bovine serum albumin.
Easily the chimeric ES cell of activated NT unit was placed the CR1aa substratum that contains 1.9mM DMAP about 4 hours, subsequently with the HECM washing, then at about 38.5 ℃ and 5%CO
2In containing the CR1aa of BSA, cultivated down about 4-5 hour.Usually wash the NT unit of cultivating, and place the appropriate condition substratum to grow.A kind of effective substratum is the CR1aa substratum that contains 10%FCS and 6mg/ml BSA.Suitable feeder layer comprises inoblast and epithelial cell, particularly uterine epithelial cell, forms the source as ungulate, chicken, mouse, STO, SI-m220 and BRL cell.Have been found that mouse embryo fibroblasts is particularly useful.(because of the species difference of nuclear) obtains chimeric ES cell behind time enough, can be used for now implanting among the suitable host.
5. the chimeric NT unit of activity is implanted in the compatible female host.
6. make chimeric NT unit be grown to embryo or mature, produce new born animal.
These stages of many species (comprising mouse and ungulate) are statement well in the literature.In order to be fertilized, NT unit is transferred in the uterus.After fertilization monitors the host, successfully implants to guarantee NT unit.According to host's character, can retrain the host, to prevent spontaneous abortion.The childbirth of tire or new born animal can be nature or artificial abortion, spontaneous labor or c-section.
7. make animal neonatal growth to a dating, produce chimeric ovocyte.
8. from the offspring, gather in the crops chimeric ovocyte.
The above-mentioned stage is depended on host's character.Normal treatment host, making animal neonatal growth is health hosts.The results of ovocyte can be carried out as mentioned above.
9. with chimeric ovocyte stoning, import and the allogenic nuclear of ovocyte plastosome, produce the chimeric NT unit of allos.
The method of stoning and importing nuclear is described.Provide the selection of the species of nuclear will depend on the purposes of cell.Cell can be used for cloning the species that specific species, particularly ovocyte are difficult to grow, and as human or rare species, perhaps as the source of noble cells, wherein cell contains nuclear and the plastosome from same species.Different with the mankind, other species that the present invention uses comprise rare and clones animals on the brink of extinction.
10. cultivate the chimeric NT unit of allos and produce the ES cell.
NT unit cultivates on feeder layer, reaches the size that is suitable for separating the ES cell up to NT unit, needs at least 4 cells usually, and preferably at least 50 cells generally are no more than 400 cells.Cultivate general use 38.5 ℃ and 5%CO
2Condition, changed substratum in about every 1-5 days.
Final stage comprises that machinery takes out NT unit from culture, from NT internal institution isolated cell, although the cell from NT unit's other parts also can use, the cell of washing NT unit, with cell inoculation in from the feeder layer of the identical or different species of nuclear on, Zhao She inoblast for example.Cell is stored in appropriate culture medium and (for example replenishes on the feeder layer of the α of 10%FCS, 0.1mM beta-mercaptoethanol and L-glutaminate-MEM).Changed growth medium in about every 1-3 days.
For human heterogenous chimeric ES cell, individual cells does not have fine definition, the border refraction and the smooth in appearance of colony.Colony does not have epithelium sample outward appearance.
Can use the NT unit cell to produce inner cell mass (ICM) cell of blastocyst.The ICM cell can utilize the feeder layer STO (l cell) of the serum of charcoal absorption (as contain) incubation growth.
11. the chimeric ES cell of genetic modification allos randomly.
The ES cell can be according to the routine techniques dna modification.Utilize suitable medium, liposome, transit sequence, penetrating, electricity merges etc., can be with the gene of the hope in DNA, virus, plasmid, YAC, exchromosomal DNA, chromosome segment, cDNA or other source of naked DNA, recA bag quilt, regulate sequence etc. and import in the ES cell.Referring to, for example, people such as Schneike, Science 278:2130-3 (1997).
Modification can provide the composing type or the inducible expression of specific product, Regular Insulin for example, angiogenesis factor, cytokine, for example interleukin, Interferon, rabbit and G CFS, blood factor, serum albumin for example, zymoplasm, Fibrinogen, thrombopoietin, erythropoietin, tissue plasminogen activator etc., hormone, for example tethelin, somatomedin, Urogastron for example, Prostatropin, glial cell derived neurotrophic growth factor etc., enzyme, enzyme inhibitors, alpha antitrypsin for example, telomere associated protein, for example Sir, Telomerase etc., neuronal protein, neurotrophic factor-3 for example, 4/5, ciliary neurotrophic factor etc., Trypsin, basic protein, taste bud albumen, eye albumen, oxyphorase, transcription factor, lipoprotein, immunoglobulin (Ig), surface film acceptor, for example insulin receptor, the oncogene inhibitor, L-Dopamine HCL and angiogenesis inhibitor.In addition, for correcting defect or undesirable phenotype, can utilize the homologous recombination of nuclear to produce the chimeric ES cell of allos.For example, can genetic modification from sicklemia patient's nuclear, obtain to produce the cell of natural hemoglobin; The haemophiliac can correct the mutator gene of blood factor (as Factor IX c or VIIIvw, Christmas); Because the easily ill patient of inherited genetic factors can modify nuclear, produce these diseases (for example AIDS, juvenile form morbidity type diabetes, muscular dystrophy, alzheimer's disease, Parkinson's disease etc.) allelotrope that susceptibility is lower.
Sometimes may not import hereditary potency, but wish to close hereditary potency or make gene become induction type.The illustrative gene that can knock out comprises that oncogene, tissue compatible protein, blood group albumen and dominance expression cause other gene of pathology.The cell that knocks out that can utilize homologous recombination or screening to wish.
12. utilize chimeric other cellular type of ES cells produce of allos.
Pedersen, Reprod.Fertil.Dev.6:543-52 (1994) has summarized a large amount of articles about the ES cytodifferentiation.The author has reported generation and the differential period or the cellular products of the noble cells of the CD marker that the express cell type is special, and cell does not produce these products with the level of ES cytodifferentiation usually.The ES cell is to the people such as the visible Bain of growth of specific cells type, Dev.Biol.168:342-357 (1995) (neurocyte); People such as Palacios, Proc.Natl.Acad.Sci.USA 92:7530-7537 (1995) (hematopoietic cell); People such as Rathjen, Reprod.Fertil.Dev.10:31-47 (1998).
The purpose cell comprises: hematopoietic cell, neuronal cell, skeletal muscle and myocardial cell, skin cells, epithelial cell, endotheliocyte, structure cell, osteoclast and scleroblast, follicular cell, eye cell, with feel relevant cell, as taste bud, interior ear cell, osteocyte, nephrocyte, liver cell, pancreatic cell, β-islet cells for example, inoblast and chondrocyte.
The ES cell of differentiation can have extensive use.The cell that contains allos plastosome and nuclear provides the normal cell that contains from the DNA of single species.Avoided the problem of uncompatibility between plastosome and the nuclear like this, the protein that exists in the tenuigenin is all from same species, nuclear and mitochondrial interaction, for example proteinic transhipment is a nature, the immundominance sequence that cell surface exists all derives from identical species.And, depend on plastosome and other interactional cell processes in intracellular region chamber also is a nature.
The cell of ES cell and differentiation provides the analysis of cells process, to many chances of the reaction of extrinsic factor (for example gene, medicine, the factor etc.), be used for screening of the effect of these extrinsic factors at drug development, determine and specific allelotrope or the sudden change relevant, study the variation of the transcript and expression that different factors cause the reaction of these external factor.
In addition, preserve also and can breed because the ES cell can be cultivated, so can repeat to produce the cell of differentiation, these cells will be based on identical genotype.The present invention also provides the ability of studying and diagnosing from the nuclear of individuality of using.Therefore, can determine the susceptibility of particular individual to external factor (as carcinogens, allergen, toxin and mutagenic compound), an intact cell of one of them species has the natural metabolism of these species.Also can study cell response, to determine to the Normocellular influence of particular patient for specified scheme (as pharmaceutical admixtures).About the chronic application of scheme, the stoning ovum that the purpose species that time enough use to store are arranged imports the nuclear from the noble cells of this species particular individual as container.Available then ES cell produces the different noble cellss that are in different differential periods, and they can accept this scheme and change as the cell that detects.
The present invention can be used to study the process of plastosome and particular core.For example, when individuality may contain the nuclear of defective aspect producing the protein that plastosome uses, this method can be used for cloning these cells, observed differentiation to the influence of plastosome process with to the influence of growth pattern, phenotype etc.
Be used for the situation that the embryo is ripe and new born animal produces in ES cell or NT unit, the uncompatibility when using natural plastosome to avoid other stud matings of the individuality of generation and same species.Therefore, owing to there is the external source mitochondrial protein, tire is less by the possibility that parent repels, and is less to the possibility of host cell immunne response.
Can be used for composition of the present invention and be separating and containing of cultivating from the plastosome of species with from the ovocyte of the nuclear of different plant species.In the time of most of, nuclear and plastosome come from the species of fundamental difference, and be general from different genus, not even Tong section.Select nuclear easily, the useful source of stoning ovum is provided, as domestic animal (ungulate), for example ox, sheep and pig, and laboratory animal are as mouse.The present invention also comprises and containing from the plastosome of body one by one with from the ovum of the nuclear of the Different Individual of same species, by the ES cell that its produces, contains the culture of these ES cells, by the noble cells of its generation with contain the culture of these noble cellss.
These compositions contain chimeric ovocyte of allos and ES cell in the grown culture that is used for cell proliferation.These compositions also comprise the mixture of allos chimeric cell and inner cell mass noble cells at the substratum that is used for cytodifferentiation, perhaps are accompanied by the growth or the preservation of ES cell and noble cells.
For hematopoietic cell, in Costar six well culture plates, a kind of substratum contains (5-10 μ g/ml, 37 ℃, 3-4 hour) or (2-4 * 10 of irradiation that ametycin is handled
3The rad gamma-rays; The RP.0.10 marrow stromal cell individual layer of 1 rad=0.01Gy) contains recombinant interleukin-3 (rIL-3) (100-300 unit/ml), rIL-6 and F (final concentration is 10%v/v), the supernatant liquor of 2 days cultures of the FLS4.1 tire liver stromal cell that is paved with.The FLS4.1 supernatant liquor contains the new factor of FLT3 part, the steel factor and a kind of hematopoiesis support stem cell growth, in 2-2.5ml substratum [Iscove ' s Dulbecco ' s improved culture medium/50 μ M 2 mercapto ethanol/2mM L-glutaminate/50 μ g/ml gentamicin/7.5%v/v FCS], 37 ℃, 7.5%CO
2/ 92.5% air.Every 5-7 days collecting cell, the cultivation of in new Costar six well culture plates of RP.0.10 stroma cell that contains the ametycin processing and freshly prepd factor-containing substratum, going down to posterity.(people such as Palacios, Proc.Natl.Acad.Sci.USA 92:7530 (1995)).Should be appreciated that and also can use other clone that similar conditioned medium is provided, and similar active other composition is provided.
For neuronal cell, the ES cell is carried out inducing program in 8 days, comprise cultivation in 4 days, become the not aggregation of retinoic acid-containing (RA), in the presence of RA, cultivated 4 days subsequently.Substratum is that DMEM (contains L-glutaminate, do not contain the high glucose of pyruvic acid; GIBCO11965-043), 10% foetal calf serum, 10% new-born calf serum and nucleosides stoste.
Other substratum also can be used for guiding to the differentiation of other cellular type, and use natural existence and influence the factor of breaking up in the body, as Hemopoietic factor, for example G-CSF, M-CSF, GM-CSF, interleukin, Interferon, rabbit and other cytokine and somatomedin.
All containing identical mitochondrial enucleation oocyte can frozen longer for some time, carefully thawing before use in appropriate culture medium.
Whether these noble cellss no matter genetic modification all can be used in treatment, and healthy cell can import and exercise the function of wishing in the suitable compartment.For example, the myocyte can be used for transplanting to cardiac muscular tissue or other muscle tissue, and the cell of natural or genetic modification may have the advantage that for example can revise mutein in muscular dystrophy.Referring to, for example, U.S. Patent number 5,602,301.Can in the patient, import hematopoietic cell, as lymphocyte, natural killer cell, megalokaryocyte, eosinophilic granulocyte, basophilic granulocyte, monocyte or its precursor, these precursors can be used for the specific cells type or can be polyenergic, and will be divided into a large amount of dissimilar cells.β-islet cells can be transplanted on the pancreas, perhaps places the protection container, and they can innervate by blood supply, are used for treatment of diabetes.Neuronal cell can import in the encephalocoele, by other factor that L-Dopamine HCL, NGE maybe can influence brain function or the source of other composition are provided, is used for the treatment of parkinsonism, alzheimer's disease, cerebral palsy etc.Other indication that ES cell or its differentiation offspring are suitable for comprises: Spinal injury, multiple sclerosis, hepatopathy, vascular disease, the displacement of burn cartilage, heart trouble, nephropathy, urinary tract disorder, prostatosis and the aging disease that causes.
These cells can be used for composing type or induction type supply specificity factor intentinonally.People such as Pruschy, Chem.Biol.1:163-72 (1997) improves, but the generation of the pill induced product by oral activating transcription factor.Therefore,, can induce to produce tissue plasminogen activator,, produce Regular Insulin for diabetes for heart attack, for infection, activated lymphocyte, method is to import the non-human acceptor, as ecdysone receptor etc.
The following example is in order to illustrate rather than to limit.
Experiment
Materials and methods
The acceptor human oocyte
Ovocyte from people's tire: behind EAB, from the fetus of 16-20 week gestation, obtain the fetus ovary.Ovary tissue is chopped into～the 1mm size, in the Waymouth substratum that has replenished 15% (v/v) foetal calf serum, 0.03 IU/ml FSH and 35ng/ml Regular Insulin, cultivates.Be organized in 37 ℃ and 5%CO
2In the Falcon plate, cultivated 5-25 days in the air, on Costar Transwell-COL film, cultivated 3-40 days, in the presence of LH and hFF, induce final maturation afterwards.The individual layer fragment of being made up of inoblast forms flesh tissue cultivating in 2-3 days.After cultivating for 1 week, ovarian follicle is told from ovary tissue, but still is attached to individual layer.The ovarian follicle of the maximum quantity of telling from tissue is beginning to cultivate about 1 week back appearance.Cultivate in the Costar plate after 40 days, the major portion diameter of ovum reaches more than 80 μ, and about 1/3rd are surrounded by zona pellucida.After inducing final maturation, in the Costar plate, observe the extruding of first polar body in 40 days the ovum of growth.Can collect sophisticated ovum, directly carry out stoning.
Ovocyte from the mature women.People's ovum utilizes people such as Trotnow, and the technology of Arch.Gynecol.236:211-7 (1985) obtains.The attraction syringe needle of trocar is passed in utilization, controls absorption with the transmodulator of DiasonicsDS 1 sector scan instrument, carries out the continuous ultrasound imaging to attracting syringe needle.The patient imposes epidural anesthesia.
Also can use other technology, comprise according to people such as Mercan, the described scheme of Hum.Reprod.12:1886-9 (1997) is used follicle-stimulating hormone.
Ovocyte then can be following at maturation in vitro.Wash immature ovocyte with the TL-HEPES buffer culture medium that contains 3mg/ml bovine serum albumin (fraction V).Ovocyte mound mixture is placed the TCM-199 that contains 10% foetal calf serum, LH and/or FSH and estradiol under 39 ℃.In maturation medium, after about 20 hours, take out ovocyte, place the TL-HEPES that contains the 1mg/ml Unidasa, repeat imbibition by the pore suction pipe and remove the mound cell.The ovocyte that screening is peeled off according to polar body, the ovocyte of selecting to contain polar body (II ovocyte in mid-term) is further used.
The ox donorcells:
The super ovulation of heifer, artificial insemination.Obtain the embryo the 5th day oestrus or the 6th day from reproductive tract.Wash the embryo with PBS from reproductive tract, and reclaim.Embryo in maturation in vitro, fertilization and cultivation was used as the donor embryo on the 5th day at after fertilization.People such as insemination process such as Keefer, Mol.Reprod.Dev.36:469-74 (1993) is described.
Stoning:
Stoning (hpm) after maturation begins about 18 hours utilizes the oblique angle micropipet to carry out.Stoning adds confirmation in the bisbentiamin (Hoechst 33342,3 μ g/ml) at the TL-HEPES substratum.
Nuclear transplantation:
Individual donorcells is placed the ovum week crack of acceptor enucleation oocyte.Human oocyte tenuigenin and ox donor nuclei (NT unit) utilize electric integration technology to merge: 90V merges pulse 25 microseconds after oocyte maturation begins 24 hours.Place the CR1aa substratum to begin back 28 hours up to maturation the NT unit that obtains, this moment, they activated according to following method.NT unit is exposed to ionomycin-6-DMAP (2mM) 4 minutes, in having replenished the sero-abluminous TL-HEPES of 1mg/ml only in DMAP (2mM) 4 hours subsequently, washs 5 minutes with adding the sero-abluminous TL-HEPES of 30mg/ml then.A droplet CR1aa substratum of then NT unit being transferred on 4 well culture plates adds in 10%FCS and the 6mg/ml serum albumin, wherein contains the feeder layer of the mouse embryo fibroblasts that is paved with.NT unit is at 38.5 ℃ and 5%CO
2Under cultivate more than 3 days.Changed a subculture in per 3 days, up to activating beginning back the 12nd day.Should form about 50 cells this moment, and machinery takes out these cells from zona pellucida, is used to produce embryo cell line.
The mouse embryo fibroblasts feeder layer:
The primary culture of mouse embryo fibroblasts obtains from 14-16 days mouse tire.Behind aseptic removal head, liver, heart and digestive tube, with embryo's chopping, and at pre-warm trypsinase EDTA solution (0.05% trypsinase/0.02%EDTA; GIBCO, Grand Island, NY) in 37 ℃ of incubations 30 minutes.Inoblast is inoculated in the tissue culture flasks, α-MEM substratum of adding 10%FCS, penicillin (100IU/ml) and Streptomycin sulphate (50 μ l/ml) (Bio Whittaker, Walkersville, MD) the middle cultivation.After going down to posterity 3-4 days, irradiation 35 * 10Nunc culture dish (Baxter Scientific, McGaw Park, IL) embryo fibroblast in.The inoblast of irradiation is containing 5%CO
2Damp atmosphere in 37 ℃ of growths and preserve.The culture plate that contains the homogeneous cell monolayer is used for cultivating embryo cell line.
The generation of embryo cell line:
The NT unit cell that washing obtains as mentioned above, direct inoculation is on the raising inoblast of irradiation (on seeing).These cells comprise the inside cell of NT unit.Cell is stored in α-MEM growth medium of adding 10%FCS and 0.1mM beta-mercaptoethanol.Changed a subculture in every 2-3 days.Observed initial colony in the 2nd day or the 3rd day to cultivating.This colony propagation shows that doing (ES) cell with previously disclosed mouse and ox embryo has similar morphology.Individual cells in the colony does not have fine definition, the border refrangible and the smooth in appearance of colony.These cells have the epithelium outward appearance.
The generation of chimeric ox NT unit:
Select chimeric NT unit, implant in the cow according to following method: 1-5 NT unit implanted in the uterus.The calf that produces grows into the suitable stage that ovocyte is grown, and sucking-off this moment also separates ovum.In addition, also can obtain from adult female by the ovocyte of super ovulation and ultrasonic guidance or from the cow ovary of slaughtering, obtain ovocyte.
The generation of the chimeric people's cell of allos:
The chimeric ovum of Huo Deing prepares to be used for receiver's nuclear then as above for growth and the described processing of stoning as mentioned above.In the one-tenth human oral of informed consent, scrape the human epithelial cell with the standard slide glass gently.Cell is flushed to the culture dish of the PBS that contains Ca or Mg from slide glass.Draw cell by small-bore suction pipe, cell lump is broken for single cell suspension.Then cell transfer is contained in the TL-HEPES substratum of 10%FCS to a droplet that is covered under the oil, utilize aforesaid method in the chimeric bovine oocyte of the stoning that contains human mitochondrion, to carry out nuclear transplantation.
The allos chimeric cell can use from the nuclear in all human cells that are fit to sources basically, as inoblast, epithelial cell, keratinocyte, blood lymphocytes or Urothelial Cell.Allos chimeric cell incubation growth, according to different differentiation models, as described in people such as Stice, (1998) with above, comprise myocyte's preparation.
According to the present invention, provide and contained from the people's nucleoid of Different Individual and the chimeric people's cell of allos of human mitochondrial.These cells have extensive use, because they provide the simulating nature human cell, are used for the cell processes of studying fetal development and breaking up to the noble cells type.These cells also can be used to produce the cell of differentiation, are used for cell therapy or produce the human factor.These cells are only to produce the human protein, so that the cell of unlikely induce immune response, permission is to the genetic manipulation of nuclear, the individuality that can import as the nuclear source is provided, but in order to treat or other purposes is examined adorned human cell, and the source of the identical cell of genotype is provided, and they can be used for the clone of specific gene type.
The reference of quoting in the application's book is incorporated herein by reference.Comprise described all programs and method, constitute the method part of the application's book,, be applicable to the theme of the application's book according to those skilled in the art.
The present invention now describes fully, it will be appreciated by those skilled in the art that, under the situation of the spirit or scope that do not deviate from the accessory claim book, can carry out many changes and modification to it.
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.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US19740700P | 2000-04-14 | 2000-04-14 | |
| US60/197,407 | 2000-04-14 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN01808875A Division CN1427887A (en) | 2000-04-14 | 2001-04-16 | Pluripotent cells comprising allogenic nucleus and mitochondria |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1673361A true CN1673361A (en) | 2005-09-28 |
Family
ID=22729292
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN01808875A Pending CN1427887A (en) | 2000-04-14 | 2001-04-16 | Pluripotent cells comprising allogenic nucleus and mitochondria |
| CNA2005100527010A Pending CN1673361A (en) | 2000-04-14 | 2001-04-16 | Multipotential cell containing heterogenous nuclear and mitochondrion |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN01808875A Pending CN1427887A (en) | 2000-04-14 | 2001-04-16 | Pluripotent cells comprising allogenic nucleus and mitochondria |
Country Status (10)
| Country | Link |
|---|---|
| EP (1) | EP1282685A2 (en) |
| JP (1) | JP2003530848A (en) |
| CN (2) | CN1427887A (en) |
| AU (1) | AU2001257053A1 (en) |
| BR (1) | BR0110072A (en) |
| CA (1) | CA2405555A1 (en) |
| IL (1) | IL152064A0 (en) |
| MX (1) | MXPA02010075A (en) |
| NZ (1) | NZ521711A (en) |
| WO (1) | WO2001079445A2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350479A (en) * | 2011-04-14 | 2017-01-25 | 通用医疗公司 | Composition and method for energy transfer of autologous germline mitochondria |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1373474A2 (en) | 2001-01-02 | 2004-01-02 | Stemron, Inc. | A method for producing a population of homozygous stem cells having a pre-selected immunophenotype and/or genotype |
| WO2003072708A2 (en) * | 2002-02-21 | 2003-09-04 | Advanced Cell Technology, Inc. | Pluripotent cells comprising allogenic nucleus and mitochondria |
| CN104830777A (en) * | 2014-02-28 | 2015-08-12 | 复旦大学 | Polar body genome reconstruction ovum, preparation method and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6011197A (en) * | 1997-03-06 | 2000-01-04 | Infigen, Inc. | Method of cloning bovines using reprogrammed non-embryonic bovine cells |
-
2001
- 2001-04-16 NZ NZ521711A patent/NZ521711A/en unknown
- 2001-04-16 CN CN01808875A patent/CN1427887A/en active Pending
- 2001-04-16 BR BR0110072-6A patent/BR0110072A/en not_active IP Right Cessation
- 2001-04-16 JP JP2001577429A patent/JP2003530848A/en active Pending
- 2001-04-16 CN CNA2005100527010A patent/CN1673361A/en active Pending
- 2001-04-16 MX MXPA02010075A patent/MXPA02010075A/en not_active Application Discontinuation
- 2001-04-16 AU AU2001257053A patent/AU2001257053A1/en not_active Abandoned
- 2001-04-16 CA CA002405555A patent/CA2405555A1/en not_active Abandoned
- 2001-04-16 WO PCT/US2001/012265 patent/WO2001079445A2/en active IP Right Grant
- 2001-04-16 IL IL15206401A patent/IL152064A0/en unknown
- 2001-04-16 EP EP01930525A patent/EP1282685A2/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350479A (en) * | 2011-04-14 | 2017-01-25 | 通用医疗公司 | Composition and method for energy transfer of autologous germline mitochondria |
Also Published As
| Publication number | Publication date |
|---|---|
| MXPA02010075A (en) | 2003-02-12 |
| WO2001079445A2 (en) | 2001-10-25 |
| CN1427887A (en) | 2003-07-02 |
| CA2405555A1 (en) | 2001-10-25 |
| BR0110072A (en) | 2004-07-06 |
| EP1282685A2 (en) | 2003-02-12 |
| AU2001257053A1 (en) | 2001-10-30 |
| NZ521711A (en) | 2005-03-24 |
| IL152064A0 (en) | 2003-05-29 |
| WO2001079445A3 (en) | 2002-07-11 |
| JP2003530848A (en) | 2003-10-21 |
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