JP2002537803A - Embryonic or stem-like cell lines produced by xenogeneic nuclear transfer - Google Patents

Abstract

  (57) [Summary] An improved method of nuclear injection is provided wherein the nuclei of the differentiated donor cells are transplanted into enucleated oocytes of an animal species different from the donor cells. The nuclear injection unit thus obtained is useful for producing syngeneic embryonic stem cells, particularly human syngeneic embryonic cells or stem cells. These embryonic or stem-like cells are used for the production of differentiated cells of interest, the introduction, removal or modification of the gene of interest (eg, at a specific location in the genome of such cells by homologous recombination). Useful. These cells may contain heterologous genes and are particularly useful for in vitro studies on cell transplantation therapy and cell differentiation. Also provided are methods of increasing nuclear injection efficiency with genetically modified donor cells to inhibit apoptosis, select a particular cell cycle, and / or promote embryo growth and development.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application is a continuation-in-part of application Ser. No. 09 / 030,945 filed on Mar. 2, 1998, and is filed on Aug. 19, 1996. It is also a continuation-in-part of the application number 08 / 699,040. The present application is fully incorporated by reference herein.

FIELD OF THE INVENTION [0002] The present invention relates to embryonic cells or stem-like cells obtained by transferring cell nuclei obtained from animal or human cells into enucleated oocytes of an animal species different from that of the donor. It concerns cell production. More specifically, the invention relates to animal oocytes enucleated from nuclei of primate or human cells, such as primate or ungulate oocytes, more preferably bovine enucleated oocytes. And the production of primate or human embryonic cells or stem-like cells by transplanting the cells into the primate.

Further, the present invention relates to the application for treatment and diagnosis of the embryonic cells or stem-like cells thus obtained, preferably primate or human embryonic cells or stem-like cells, and the therapeutic or diagnostic method. For use in the production of transgenic embryonic cells or transgenic differentiated cells, cell lines, tissues and organs. Further, the embryonic cells or stem-like cells produced according to the present invention may be used as a nuclear donor in a nuclear transfer or nuclear injection method for producing a chimera or clone, preferably a transgenic cloned animal or chimeric animal. Is also possible.

BACKGROUND OF THE INVENTION [0004] Methods for establishing in vitro embryonic stem (ES) cell lines from preimplantation early embryos of mice are well known. (See, for example, Evans et al., Nature, 29: 154-156.
(1981); Martin, Proc. Natl. Acad. Sci., USA, 78: 7634-7638 (1981). ES cells are undifferentiated in the presence of fibroblast feeder layers (Evans et al., Supra) or differentiation inhibitors (Smith et al., Dev. Biol., 121: 1-9 (1987)). It is possible to passage in the state.

[0005] ES cells have been reported to have various fields of application. For example,
It has been reported that ES cells can be used as a model for differentiation in vitro, particularly for studies of genes involved in early developmental regulation. Mouse ES cells grow into germline chimeras when introduced into mouse preimplantation embryos,
Pluripotency has been confirmed (Bradley et al., Nature, 309: 255-256 (1984)).
.

[0006] Judging from the ability of ES cells to transmit their genome to the next generation, the ES cells can be used for livestock animals, whether or not genetic modification suitable for the purpose is performed. It has the utility that germline manipulation is possible. Furthermore, in the case of livestock animals such as ungulates, the removal of nuclei from preimplantation embryos of livestock helps the enucleated oocytes to develop to birth (Smith et al., Biol.
Reprod., 40: 1027-1035 (1989); and Keefer et al., Biol. Reprod., 50: 9.
35-939 (1994)). This is in contrast to nuclei from mouse embryos, which have been reported to not support the development of enucleated oocytes when injected after the 8-cell stage (Ch
eong et al., Biol. Reprod., 48: 958 (1993)). Thus, from livestock animals
ES cells are very promising because they are likely to be the source of totipotent donor nuclei for nuclear injection, with or without genetic manipulation.

[0007] Several research groups have reported the isolation of a putative pluripotent embryonic cell line. For example, Notarianni et al., J. Reprod. Fert. Suppl., 43: 255-260.
(1991) established a cell line presumed to be stable and pluripotent from undifferentiated germ cells of pigs and sheep, and during primary culture of inner cell mass isolated from undifferentiated germ cells of sheep by immunosurgical means. Exhibit some morphological and growth characteristics similar to the cells in Id. Notarianni et al., J, Reprod. Fert. Suppl
, 41: 51-56 (1990) also disclose the maintenance and differentiation of putative pluripotent embryonic cell lines from porcine undifferentiated germ cells in culture. In addition, Gerfen et al.
Anim. Biotech, 6 (1): 1-14 (1995) disclose the isolation of embryonic cell lines from porcine undifferentiated germ cells. These cells can be stably maintained in the mouse embryonic fibroblast feeder layer without using conditioned media. These cells have been reported to differentiate into several cell types in culture (Gerfen
et al., ibid.).

Further, Saito et al., Roux's Arch. Dev. Biol., 201: 134-141 (1992)
It has been reported that when a bovine embryonic stem-like cell line was cultured, it survived to the third generation but died after the fourth generation. Further, Handyside et al., Roux's Arch. Dev. Biol., 1
96: 185-190 (1987) discloses the cultivation of sheep embryonic inner cell mass isolated by immunosurgical techniques under the same conditions as for isolating mouse ES cell lines from mouse ICM. Handyside et al. (1987) (supra) show that under these conditions sheep ICM attaches, spreads, and forms regions of both ES-like and endoderm-like cells, but when cultured for long periods, It is reported that only germ layer-like cells are observed (ibid.).

Recently, Cherny et al., Theriogenology, 41: 175 (1994) reported that a cell line derived from putative bovine primordial germ cells could be maintained in long-term culture.
After culturing for about 7 days, these cells produced colonies that stained for alkaline phosphatase (AP) positive, showed the ability to form embryoid bodies, and spontaneously differentiated into at least two cell types. These cells also have mRNAs for the transcription factors OCT4, OCT6 and HES1, a pattern of homeobox genes that are thought to be expressed only by ES cells.
Has been reported to be expressed.

[0010] More recently, Campbell et al., Nature, 380: 64-68 (1996) reported a culture from sheep day 9 embryos cultured under conditions that facilitated the isolation of ES cell lines in mice. We reported that lambs were born after nuclear injection of embryonic disc (ED) cells. Based on his results, the author concluded that ED cells from sheep day 9 embryos were totipotent by nuclear injection and that totipotency was maintained in culture.

[0011] Van Stekelenburg-Hamers et al., Mol. Reprod. Dev., 40: 444-454 (1995
) Reported the isolation and characterization of putative permanent cell lines established from inner cell mass cells of bovine undifferentiated germ cells. The authors isolated and cultured bovine ICM from bovine undifferentiated germ cells on day 8 or day 9 under various conditions.
We examined which feeder layers and media were most effective in supporting cell attachment and growth. Based on their results, STO as an additive to the culture medium
It was concluded that the use of (mouse fibroblasts) feeder cells (instead of bovine uterine epithelial cells) and charcoal-filtered serum (rather than normal serum) promoted attachment and growth of cultured ICM cells. However, Van Stekelenburg et al. Reported that their cell lines were more like epithelial cells than pluripotent ICM cells.

Further, Smith et al., WO 94/24274, published Oct. 27, 1994, Evans et al., 19
WO 90/03432, published on April 5, 1990, and WO 94, published on November 24, 1994, by Wheeler et al.
/ 26889 report on the isolation, selection and expansion of putative animal stem cells that can be used to produce transgenic animals. Evans et al.,
WO 90/03432, published April 5, 1990, reports the production of putative pluripotent embryonic stem cells from porcine and bovine species and claims to be useful for the production of transgenic animals. ing. Further, Wheeler et al., Published on November 24, 1994, WO
94/26884 published embryonic stem cells allegedly useful for the production of chimeric and transgenic ungulates. Thus, based on the above examples, many groups have attempted to produce ES cell lines because ES cell lines have potential for application in the field of clonal or transgenic embryo production and nuclear transfer. It is clear that

[0013] The use of ungulate ICM cells for nuclear transfer has also been reported. For example, Col
las et al., Mol. Reprod. Dev., 38: 264-267 (1994) discloses bovine IC by microinjecting lysed donor cells into enucleated mature oocytes.
The M port is open to the public. In this document, embryos were cultured in vitro for 7 days
It has been published that four undifferentiated germ cells were produced and transplanted into bovine recipient animals, resulting in the pregnancy of four animals and the birth of two animals. Also, Keefer et al.,
Biol. Reprod., 50: 935-939 (1994) discloses that several calves were born using bovine ICM cells as donor nuclei in a nuclear transfer procedure. In addition, Sims
Natl. Acad. Sci, USA, 90: 6143-6147 (1993) describe the transfer of nuclei from bovine ICM cells cultured in vitro for short periods to enucleated mature oocytes. The calf was born.

It has also been reported that lambs were born after nuclear transfer of cultured embryonic disc cells (Campbell et al., Nature, 380: 64-68 (1996)). further,
It has also been reported that bovine pluripotent embryonic cells were used in nuclear transfer to produce chimeric fetuses (Stice et al., Biol. Reprod., 54: 100-110 (1996); Collas et al.).
al., Mol. Reprod. Dev., 38: 264-267 (1994).

Attempts have been made to create a heterogeneous NT unit (Wo).
lfe et al., Theriogenology, 33: 350 (1990). Specifically, bovine embryonic cells were fused with wild bovine oocytes to produce xenogeneic NT units that could have an inner cell mass. However, embryonic cells rather than adult cells were used as donor nuclei in the nuclear transfer method. The claim was that embryonic cells could be reprogrammed more easily than adult cells. This dates back to earlier studies of NT in frogs (reviewed by DiBerardino, Differentiation, 17: 17-30 (1980)). The study also used animals that were very phylogenetically similar (bovine nuclei and wild bovine oocytes). In contrast, previous fusions between NTs in more diverse animal species (bovine nuclei into hamster oocytes) did not yield the structure of the inner cell mass. Furthermore, it has not been reported that an ES cell-like colony capable of growing using the inner cell mass cells from the NT unit was formed.

Collas et al. (Ibid.) Also suggested using granulosa cells (mature somatic cells) to produce bovine nuclear transfer embryos. However, unlike the present invention, these experiments did not perform xenotransplantation. Also, unlike the present invention, no ES cell-like colonies were obtained.

Very recently, published on December 1, 1998 by James A. Thomson, Wisconsin Alumni Re
U.S. Patent No. 5,843,780, assigned to the search Foundation, claims to disclose the purification and preparation of primate embryonic stem cells having the following characteristics:
i) can proliferate for more than one year in in vitro culture; (ii) have a karyotype that has all chromosomes characteristic of primates and does not change significantly even after long-term culture. (Iii) maintain the ability to differentiate into endoderm, mesoderm and ectoderm tissue derived organs throughout the culture period; and (iv) when cultured on fibroblast feeder layers Does not differentiate into These cells contain the presence of all chromosomes that are SSEA-1 marker negative, SEA-3 marker positive, SSEA-4 marker positive, show alkaline phosphatase activity, are pluripotent, and are characteristic of primates It has been reported that the chromosome being used has no karyotype.
In addition, these cells are positive for TRA-1-60 and TRA-1-81 markers. Cells are claimed to differentiate into endoderm, mesoderm and ectoderm cells when injected into SCID mice. Also, an embryonic stem cell line allegedly derived from human or primate undifferentiated germ cells has been described by Thomson et al., Science 282: 1145.
Natl. Acad. Sci., USA 92: 7844-7848 (1995).

Thus, Thomson discloses cells claimed to be non-human primates and human embryonic or stem cells and methods for producing them. However, in order to enhance the therapeutic and diagnostic effects, there is a great need for a method for producing autologous human embryonic cells or stem-like cells for recipients to be transplanted.

In this regard, a number of human diseases that may be treatable by cell transplantation have been identified. For example, Parkinson's disease is caused by degeneration of dopaminergic neurons in the substantia nigra. The standard treatment for Parkinson's disease is the administration of L-DOPA,
This temporarily improves the disappearance of dopamine, but has severe side effects,
After all, the progress of the disease cannot be stopped. Another approach to the treatment of Parkinson's disease, promised to have a wide range of applications for the treatment of many brain diseases and central nervous system injuries, is to transfer cells or tissues from an animal fetus or neonate to the adult brain. It is a method of transplanting to. Fetal neurons from various parts of the brain can be taken into the adult brain. With such transplantation in laboratory animals,
Experimentally induced behavioral deficits, including complex cognitive functions, have been shown to be reduced. Initial results from human trials were also promising. But,
The supply of human fetal cells or tissues obtained by abortion is very limited. In addition, harvesting cells or tissues from aborted fetuses is very controversial.

At present, there is no means for producing “fetal-like” cells from a patient. In addition, obtaining tissue for allograft is not easy and transplant rejection occurs in both allografts and xenografts. Further, in some cases, it may be more useful to perform genetic modification on cells or tissues before transplantation. However, many of the cells or tissues for which such modifications would be desirable do not divide well in culture, and most types of genetic transformation require actively dividing cells.

Thus, the supply of transplanted tissues and cells, and human embryonic or stem-like cells for use in gene therapy, has a clear need in the art.

Objects of the Invention It is an object of the present invention to provide a new and improved method for producing embryonic or stem-like cells.

A more specific object of the present invention is to produce embryonic or stem-like cells for use in transplanting mammalian or human cell nuclei into enucleated oocytes of another animal species. To provide a new way.

[0024] Another specific object of the present invention is to provide an animal or human oocyte that has enucleated the nucleus of a non-human primate or human cell (eg, an ungulate, human or primate enucleated). The present invention provides a new method for producing non-human primate or human embryonic or stem-like cells for use in transplantation into oocytes.

Another object of the present invention is to provide a method for transplanting a nucleus of a non-human primate or human cell, for example, transplanting an adult human cell into a denucleated non-human primate or human oocyte. To provide a new method for producing non-human primate or human lineage-defective embryonic or stem-like cells for use in such cells, such that such cells cannot be differentiated into specific cell lineages Or has been modified to "kill" the cell so that it does not produce live progeny, for example, by manipulating the expression of an antisense or ribozyme telomerase gene.

[0026] Yet another object of the present invention is to provide a donor somatic cell for use in nuclear transfer to express a gene that promotes embryonic development (eg, the MHC 1 family, and particularly the Ped gene such as Q7 and / or Q9). To improve the efficiency of nuclear transfer by performing genetic manipulation, specifically, to promote the development of pre-implantation embryos produced by nuclear transfer.

It is yet another object of the present invention to enhance the production of embryos for nuclear transfer by IVP, and more specifically, for example, genes that inhibit apoptosis (eg, Bcl-2 or Bcl
Donor cells for use in nuclear transfer by introducing a DNA construct that expresses the C.-2 family) and / or by expressing an antisense ribozyme specific for the gene that induces apoptosis during early embryonic development. Genetic engineering so as to be resistant to apoptosis to promote the production of embryos for nuclear transfer.

It is a further object of the present invention to provide a DNA construct encoding a particular cyclin that binds to a marker detectable by a donor cell (eg, a DNA encoding a visually observable (eg, fluorescently labeled) marker protein). Is to increase the efficiency of selection of donor cells in a specific cell phase (for example, GI phase) by performing genetic manipulation to express, and to increase the efficiency of nuclear transfer.

Another object of the present invention is to culture embryos produced in vitro in the presence of one or more protease inhibitors, preferably one or more capsase inhibitors, to inhibit apoptosis. To promote embryonic development.

It is another object of the present invention to provide embryonic or stem-like cells produced by implanting animal or human cell nuclei into enucleated oocytes of different animal tumors.

A more specific object of the present invention is to implant into primate or human oocyte denucleated human cell nuclei (eg, human, primate or ungulate enucleated oocytes). To supply primate or human embryonic or stem-like cells to be produced.

Another object of the present invention is to use such embryonic cells or stem-like cells for treatment or diagnosis.

A specific object of the present invention is to provide such primate or human embryonic cells or stem-like cells for the treatment or diagnosis of any disease for which cell, tissue or organ transplantation is of therapeutic or diagnostic benefit. The use of cells.

Another specific object of the present invention is to use embryonic or stem-like cells produced according to the present invention to produce differentiated cells, tissues or organs.

A further specific object of the present invention is to use primate or human embryonic cells or stem-like cells produced according to the invention for the production of differentiated cells, tissues or organs. .

Another specific object of the present invention is to use embryonic or stem-like cells produced according to the present invention to produce genetically engineered embryonic or stem-like cells. Indeed, these cells may be used to produce engineered or transgenic human differentiated cells, tissues or organs (eg, used in gene therapy).

Another specific object of the present invention is to convert embryonic or stem-like cells produced according to the invention in vitro, for example for the study of cell differentiation and for assay purposes, for example for drug testing. Is to use it.

Another object of the present invention is to provide an improved transplantation therapy comprising utilizing isogenic or syngenic cells, tissues or organs produced from embryonic or stem-like cells produced according to the invention. Is to provide the law. Such treatments include Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, spinal cord injury, multiple sclerosis, muscular dystrophy, diabetes, liver disease, heart disease, cartilage replacement,
Treatment of diseases and injuries such as burns, vascular diseases, urinary tract diseases, and immunodeficiency, bone marrow transplantation,
Treatment of cancer and other diseases is included by way of example.

Another object of the present invention is to provide transgenic or genetically engineered embryonic cells produced according to the invention for gene therapy, in particular for the treatment and / or prevention of the abovementioned diseases and injuries or The use of stem-like cells.

Another object of the present invention is to provide an embryonic cell or stem-like cell produced according to the present invention,
Alternatively, use of a transgenic or genetically engineered embryonic or stem-like cell produced according to the present invention as a nuclear donor for nuclear transfer.

It is yet another object of the present invention to provide a genetically engineered ES cell produced according to the present invention to produce a transgenic animal (eg, a non-human primate, rodent, ungulate, etc.). Is to use. Such transgenic animals
It can be used, for example, for the production of animal models for the study of human diseases or for the production of polypeptides which are adapted for therapeutic or nutritional pharmaceutical purposes, for example.

With the foregoing and other objects, the advantages and features of the present invention will become apparent in the future, reference will now be made to the following detailed description of preferred embodiments of the invention and to the appended claims. This will allow a clearer understanding of the essence of the present invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides for the production of embryonic or stem-like cells, more specifically, non-human primate or human embryonic or stem-like cells by nuclear injection or nuclear transfer. To provide a new way to In this application, the terms nuclear injection or nuclear transfer or NT are used synonymously.

As described above, no actual isolation of embryonic cells or stem-like cells by nuclear injection or nuclear transfer has been reported so far. Previously reported isolation of ES-like cells was from fertilized embryonic. Also, cells or DNA of genetically different animal species, more specifically adult cells or DNA of one species (eg, human)
There have been no reports of successful nuclear transfer using oocytes from another unrelated animal species. ES cells were not obtained when embryogenesis was produced by cell fusion between closely related species such as, for example, cow-goat and cow-cattle (Wolfe et al., Theriogeno).
logy, 33 (1): 350 (1990)). Also, no method has been reported for producing primate or human ES cells from non-fetal tissue material. The limited human fetal cells and tissues currently available must be obtained and sourced from spontaneously aborted tissues and aborted fetuses.

Prior to the present invention, no one obtained embryonic cells or stem-like cells by nuclear transfer between different species.

Quite unexpectedly, the inventors of the present invention have shown that human embryonic cells, such as adult differentiated human cells, can be transplanted into enucleated animal oocytes by transplanting the nuclei into the cells. Cells or stem-like cells and cell colonies may be obtained and can be used to produce nuclear injection units (NTs), which are cells that form human embryonic cells or stem-like cells and cell colonies when cultured. discovered. The result was very surprising. Because animal cultures that do not genetically resemble differentiated donor cells or nuclei to produce a nuclear injection unit consisting of embryonic or stem-like cells and cells that form cell colonies when cultured under appropriate conditions Effective xenotransplantation (eg, transplanting cell nuclei from differentiated animal or human cells (eg, adult cells) into enucleated eggs of different animal species) by introducing them into enucleated oocytes of This is the first example confirmed.

The NT units used to produce ES-like cells may be up to a minimum of 2 to 400 cells, preferably up to 4 to 128 cells, and most preferably at least about 5
It is desirable to be able to culture until there are no cells.

In the present invention, embryonic cells or stem-like cells refer to cells produced according to the present invention. Because of the typical method for producing them, ie, xenogeneic nuclear transfer, the present application refers to such cells as “stem-like cells” rather than “stem cells”. These cells are expected to have similar differentiation potential as normal stem cells, but may have minor differences due to the method of their production. For example, these stem-like cells may have the mitochondria of the oocyte used for nuclear transfer and may not behave the same as the original embryonic stem cells.

The present invention provides a method for culturing a human adult cell nucleus when nuclear transplantation is performed, specifically, when human epithelial cells collected from the oral cavity of a human donor are transplanted into enucleated bovine oocytes. It was based on the finding that a nuclear injection unit, which is a cell forming a human stem-like cell or embryonic cell and a human embryonic cell or stem-like cell colony, was formed. This result was reproduced by the recent successful production of undifferentiated germ cells and ES cell lines as a result of transplanting human adult keratinocytes into enucleated bovine oocytes. Based on this, bovine and human oocytes, and mammalian oocytes in general, need to go through a maturation process during embryonic development, because bovine oocytes are human oocytes. The inventors have hypothesized that they are sufficiently similar or conserved that they can function as effective surrogates or substitutes for cells. Obviously, oocytes are composed entirely of proteins or nucleic acid-based factors and, under appropriate conditions, induce embryonic development, and their functions are identical or very similar in different species. Similar. These factors include material RNA
And / or telomerase may be included.

Based on the fact that human cell nuclei can be efficiently implanted into bovine oocytes, human cells can be transferred to other unrelated animal species, such as other ungulates and other animals. It makes sense to expect that they could be implanted into oocytes. In particular, other ungulate oocytes, such as pigs, sheep, horses, goats, etc., should be suitable. Also, for example, other primates, amphibians, rodents, rabbits,
Oocytes from other sources should be suitable, such as oocytes from guinea pigs and the like. Furthermore, it should be possible to transplant human cells or cell nuclei into human oocytes using the same method, and to produce human ES cells using the obtained undifferentiated germ cells.

Thus, among the most broad aspects of the present invention are animal or human cell nuclei or enucleated oocytes of animal species that differ from donor nuclei by injection or fusion of animal or human cells. To produce NT units containing embryonic or stem-like cells and / or cells that can be used to obtain cell culture. For example, the present invention provides for the transfer of ungulate cell nuclei or ungulate cells into enucleated oocytes of another animal species (eg, another ungulate or non-ungulate) by injection or fusion. Concatenating those cells and / or nuclei to produce NT units, and multicellular NT units (preferably at least about 2-400 cells, more preferably 4-128 cells, and most preferably at least about Culturing under appropriate conditions to obtain 50 cells (containing 50 cells). Such NT unit cells can be cultured and used to produce embryonic cells or stem-like cells or cell colonies.

However, in a preferred embodiment of the present invention, oocytes of human, primate, or non-primate animals (eg, ungulate oocytes) in which the nucleus of a donor human cell or a human cell is enucleated And in a preferred embodiment, a method of producing non-human primate or human embryonic or stem-like cells by transplantation into bovine enucleated oocytes.

In general, embryonic or stem-like cells are produced by a nuclear injection process consisting of the following steps: (i) harvesting the desired human or animal cells to be used as donor nucleus material ( (Ii) the oocyte may be obtained from a suitable material, such as a mammal, most preferably a primate or ungulate, such as a cow, or (iii) the egg. (Iv) injecting human or animal cells or nuclei into enucleated oocytes of an animal species different from the donor cells or nuclei, for example by fusion or injection; (Vi) culturing the cells obtained from the embryo to obtain embryonic cells or stem-like cells and stem-like cell colonies.

Nuclear injection or nuclear transfer techniques are known in the literature and are described in many of the references cited in the “Background of the Invention” section. In particular, Campbell et al., Theriogen
ology, 43: 181 (1995); Collas et al., Mol. Report Dev., 38: 264-267 (1
994); Keefer et al., Biol. Reprod., 50: 935-939 (1994); Sims et al.,
Natl. Acad. Sci., USA, 90: 6143-6147 (1993); WO 94/26884; WO 94.
/ 24274, and WO 90/03432. These are fully incorporated by reference herein. Also, U.S. Patent Nos. 4,944,384 and 5,057,420
Describe a procedure for bovine nuclear transfer. Cibelli et al., Science
280: 1256-1258 (1998).

Human or animal cells, preferably mammalian cells, can be obtained and cultured by well-known methods. Human and animal cells useful in the present invention include, for example, epithelial nerve cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), other immune cells, erythrocytes, macrophages , Melanocytes, monocytes, monocytes, fibroblasts, cardiomyocytes,
And other muscle cells. In addition, human cells used for nuclear injection include various organs such as skin, lung, pancreas, liver, stomach, intestine, heart, genitalia, bladder, kidney,
It can be collected from the urethra and other urinary tract organs. These are just a few examples of suitable donor cells. Suitable donor cells, ie, cells useful in the present invention, can be obtained from any cell or organ in the body.
That is, all somatic or germ cells are included. Desirably, the donor cell or nucleus is composed of actively dividing, ie, non-quiescent, cells. This is because it has been reported that it increases the cloning efficiency. Preferably, such donor cells are in the G1 cell phase.

The undifferentiated germinal cells obtained in this way are fully incorporated by reference herein, Thomson et al., Science 282: 1145-1147 (1998) and T.
Natl. Acad. Sci., USA 92: 7544-7848 (1995) and can be used to obtain embryonic stem cell lines based on the culture method reported by homson et al.

In the examples given below, the cells used as donors for nuclear transfer were epithelial cells from the oral cavity of a human donor and keratinocytes of a human adult. However, as discussed above, the disclosed methods can be applied to other human cells or nuclei. In addition, cell nuclei can be obtained from both human somatic and germ cells.

[0058] The donor cells can also be arrested at mitosis prior to nuclear transfer using appropriate techniques known in the art. A detailed review of methods for arresting the cell cycle at each phase is provided in U.S. Pat. No. 5,262,409, which is fully incorporated by reference herein. In particular, cyclohexamide has been reported to have an inhibitory effect on division (Bowen and Wilson (
1955) J. Heredity 45: 3-9) can be used to promote activation of mature bovine follicles when combined with electropulse therapy (Yang et al. (1992)
) Biol. Reprod. 42 (Supplement 1): 117).

Activation of the zygote gene is associated with histone H4 hyperacetylation. Trichostatin A, like other substances, has been shown to reversibly inhibit histone deacetylase (Adenot et al., 1 cell after specific H4 acetylation of male and female chromatin occurs). Mouse embryo DNA replication and pronuclear specific transcriptional activity are observed Development (November 1997) 124 (22): 4615-4625;
al., Trichostatin A and trapoxin: new chemical probes for the role of histone acetylation in chromatin structure and function. Bioessays (May 1995) 17 (5): 423-430).

As an example, butyrate is also believed to cause histone hyperacetylation by inhibiting histone deacetylase. In general, butyrate is thought to regulate gene expression, and in most cases, the addition of butyrate during culture appears to arrest cell growth. The use of butyrate in this regard is described in U.S. Patent No. 5,681,718 and is incorporated by reference herein. Thus, the donor cells can be exposed to trichostatin A or another suitable deacetylase inhibitor prior to fusion, or such substances can be added to the culture prior to genomic activation.

[0061] It is also thought that DNA needs to be demethylated in order for transcription factors to properly access DNA regulatory sequences. It has been previously reported that pre-implantation embryos undergo global demethylation in DNA from the 8-cell stage to the undifferentiated germinal cell stage (Ste
in et al., Mol. Reprod. & Dev. 47 (4): 421-429). Also, Jaenisch et al.
(1997) may be able to utilize 5-azacytidine to reduce the level of DNA methylation in cells and increase the access of transcription factors to DNA regulatory sequences. It is reported. Thus, it is possible to expose the donor cells to 5-azacytidine (5-Aza) prior to fusion, or to add 5-Aza to the culture from the 8-cell stage to the undifferentiated germ cells. In addition, other known methods for effectively demethylating DNA can be used.

Oocytes used for nuclear injection may be obtained from animals, including mammals and amphibians. Suitable mammalian oocyte sources include sheep, cattle, sheep-like animals (ovine),
Pigs, horses, rabbits, goats, guinea pigs, mice, hamsters, rats, primates, humans and the like are included. In a preferred embodiment, the oocyte will be obtained from a primate or a host, such as a cow.

[0063] Methods for isolating oocytes are known in the art. In essence, this involves isolating the oocytes from the ovaries or genitalia of mammals or amphibians, such as cattle. A readily available source of bovine oocytes is slaughterhouse material.

To be successful using techniques such as genetic manipulation, nuclear injection, and cloning, oocytes are generally isolated before using these cells as recipient cells for nuclear injection and sperm cells It must be matured in vitro before it can fertilize and develop into an embryo. This process generally involves recovering immature (prophase I) oocytes from an animal's ovaries, such as bovine ovaries obtained at a slaughterhouse, and allowing the oocytes to undergo fertilization or enucleation. Maturation of the oocyte in the maturation medium until the metaphase II stage is reached may occur, but the stage of maturation generally occurs about 18-24 hours after aspiration for bovine oocytes. For the purposes of the present invention, this period is known as the "maturity period". As used herein with reference to the calculation of this period, the term "aspiration" refers to the aspiration of immature oocytes from follicles.

In addition, metaphase II stage oocytes matured in vivo have been used successfully in nuclear injection techniques. Essentially, mature metaphase II oocytes are surgically derived from either non-ovulatory or hyperovulatory cows or heifers 35-48 hours after infusion of human chorionic gonadotropin (hCG) or similar hormones. Was collected.

The stage of oocyte maturation during enucleation and nuclear injection has been reported to be important for the success of the NT method. (See, eg, Prather et al., Differentiation, 48, 1
-8, 1991). In general, successful mammalian embryo cloning practice has used metaphase II oocytes as recipient oocytes, for the same reason as in fertilized sperm at this stage. The oocyte can be "activated" to treat the introduced nucleus, or "activated" sufficiently.
This is because it is believed that In livestock animals, especially cattle, the activation period of the oocyte is generally about 16-52 hours, preferably about 28-42 hours after aspiration.

For example, immature oocytes were isolated from Seshagine et al., Biol. Reprod. 40, 544-60.
6, after washing in HEPES-buffered hamster embryo culture medium (HECM) and at 39 ° C under a light paraffin or silicon layer as described in 1989, luteinizing hormone (LH) and follicle stimulating hormone (FSH). 50 μg / ml of tissue culture medium (TCM) 199 containing 10% fetal calf serum containing suitable gonadotropin, as well as estradiol
To the droplets of the maturation medium consisting of

After a period of maturation, typically in the range of about 10-40 hours, preferably in the range of about 16-18 hours, the oocytes are enucleated. Prior to enucleation, oocytes are preferably harvested and added to HECM containing 1 mg / ml hyaluronidase before removing cumulus cells. This may be done by repeated pipetting with a very small diameter pipette or by brief stirring with a vortex mixer. The detached oocytes are then screened for the presence of polar bodies and the selected metaphase II oocytes determined by the presence of polar bodies are used for nuclear injection. The enucleation is as follows.

[0069] Enucleation may be performed by known methods, as described in US Pat. No. 4,994,384, which is incorporated herein by reference. For example, metaphase II oocytes were treated with HECM, 7.5 μg / ml cytochalasin B for selective, immediate enucleation.
Or to a suitable medium, such as CR1aa supplemented with 10% estrus bovine serum, and then enucleated, preferably within 24 hours,
More preferably, it may be enucleated after 16 to 18 hours.

Enucleation may be performed microsurgically using a micropipette to remove polar bodies and adjacent cytoplasm. The oocytes are then screened to identify oocytes that have been successfully enucleated. This screening may be performed by staining the oocytes with a 1 μg / ml solution of 33342 Hoechst dye in HECM, and then irradiating with ultraviolet light for less than 10 seconds to observe the oocytes. Successfully enucleated oocytes can be added to a suitable culture medium.

In the present invention, the recipient oocyte is from about 10 to about 40 after initiation of in vitro maturation.
Preferably, it is enucleated over time, more preferably about 16 to about 24 hours after the start of in vitro maturation, and most preferably about 16 to 18 hours after the start of in vitro maturation.

Typically, a single animal or human cell that is heterologous to the enucleated oocyte, or a nucleus derived therefrom, is ovulated with the enucleated oocyte used to generate NT units. Inject into the cavity. Animal or human cells or nuclei and enucleated oocytes are used to produce NT units according to methods known in the art. For example, cells
The fusion may be performed by electric fusion. Electrofusion is performed by providing sufficient electrical pulses to transiently disrupt the cytoplasmic membrane. The disruption of this cytoplasmic membrane is very short because the membrane reforms rapidly. In essence, when the two adjacent membranes are induced to break, upon reformation, the lipid bilayer mixes and
A small channel will open between the two cells. Because such a small opening is thermodynamically unstable, it expands until the two cells become one. For further discussion of this process, see US Pat. No. 4,997,384 to Prather et al., The entire text of which is incorporated herein by reference. For example, a variety of electrofusion media can be used, including sucrose, mannitol, sorbitol, and phosphate buffers. Fusion can also be performed using Sendai virus as a fusion generator (Graham, Wister Inot. Symp. Mon.
ogr. 9, 19, 1969).

Similarly, in some cases (eg, a small donor nucleus), it may be preferable to inject the nucleus directly into the oocyte rather than using electroporation fusion. Such techniques are described in Collas and Barnes, Mol, which is hereby incorporated by reference in its entirety.
Reprod. Dev. 38: 264-267 (1994).

Preferably, the human or animal cell and the oocyte are at about the onset of maturation of the oocyte.
Twenty-four hours later, by applying an electrical pulse of 90-120 V for about 15 microseconds, 5
Perform electrofusion in a 00 μm chamber. After fusion, the resulting fused NT units are
For example, add to a suitable medium identified below until activation. Typically, activation occurs shortly thereafter, typically within 24 hours, preferably after about 4-9 hours.

[0075] NT units may be activated by known methods. Such methods include, for example, culturing the NT unit at a physiological temperature, by applying a cold or actually cool temperature shock to the NT unit. This is most conveniently accomplished by culturing the NT units at room temperature, which is lower than the physiological temperature conditions to which the embryos are normally exposed.

Alternatively, activation may be performed by applying a known activator. For example, penetration of oocytes by sperm during fertilization has been shown to activate pre-fused oocytes, resulting in a number of surviving pregnancies and a number of genetically identical cows after nuclear injection. I have. Similarly, treatments such as electrical and chemical shock or cycloheximide treatment may be used to activate NT embryos after fusion as well. Suitable oocyte activation methods are described in Susko-, incorporated herein by reference.
The subject of US Pat. No. 5,496,720 to Parrish et al.

For example, activation of an oocyte may be performed by simultaneously or sequentially: (i) increasing the level of divalent cations in the oocyte; and (ii) egg Reduces phosphorylation of cellular proteins in the mother cell. This is generally a divalent cation, such as magnesium, strontium,
This may be done by introducing barium or calcium in the form of an ionophore into the cytoplasm of the oocyte. Other methods of increasing divalent cation levels include the use of electric shock, ethanol treatment and caged chelator treatment.

Phosphorylation is reduced by known methods, for example, by adding kinase inhibitors such as 6-dimethylaminopurine, staurosporine, 2-aminopurine, and serine threonine kinase inhibitors such as sphingosine. Is also good.

Alternatively, phosphorylation of cellular proteins may be inhibited by introducing phosphatases, such as phosphatase 2A and phosphatase 2B, into the oocyte.

Activated NT units may be cultured in a suitable in vitro culture medium until embryonic or stem cell-like cells and cell colonies are generated. Suitable culture media for embryo culture and maturation are well known in the art. Examples of known media that may be used for bovine embryo culture and maintenance include Ham F-10 + 10% fetal calf serum (FCS), tissue culture media-199
(TCM-199) + 10% fetal calf serum, Tyrode albumin lactate pyruvate (TALP), Dulbecco's phosphate buffered saline (PBS), Eagle and Whitten media. One of the most common media used for oocyte harvesting and maturation is TCM-199, which contains fetal calf serum, neonatal serum, estrous bovine serum, lamb serum or male calf serum. Contains 1-20% serum additive. Earl salt, 10
TCM-199 with 5% fetal calf serum, 0.2 mM sodium pyruvate, and 50 μg / ml gentamicin sulfate. Any of the above, granulosa cells, fallopian tube cells, B
It may include co-culture with various cell types such as RL cells, uterus cells and STO cells.

In particular, human endothelial cells of the endometrium are associated with leukemia inhibitory factors (before and during implantation).
LIF). Therefore, adding LIF to the culture medium can be important to enhance the in vitro development of reconstructed embryos. The use of LIF for embryo or stem cell-like cell culture is described in US Pat. No. 5,712,156, which is incorporated herein by reference.

[0082] Another maintenance medium is Rosenkrans, which is incorporated herein by reference.
It is described in U.S. Pat. No. 5,096,822 to Jr et al. This embryo medium, named CR1, contains the nutrients necessary to maintain the embryo. CR1 contains L-hemicalcium lactate in an amount of 1.0 mM to 10 mM, preferably in an amount of 1.0 mM to 5.0 mM. L-Hemicalcium lactate is L-lactate with a hemicalcium salt incorporated therein.

[0083] Similarly, suitable culture media for maintaining human embryonic cells in culture are Thomso
n et al., Science 282: 1145-1147 (1998) and Proc. Natl. Acad. Sci. USA.
92: 7844-7848 (1995).

Thereafter, the cultured NT unit or units are preferably washed, and then a suitable medium, such as CRIaa medium, preferably containing about 10% FCS, ham F-10, tissue culture medium-1
99 (TCM-199), Tyrode albumin lactate pyruvate (TALP), Dulbecco's phosphate buffered saline (PBS), Eagle's or Whitten's medium. Such culture is preferably performed in a well plate containing a suitable confluent feeder layer. Suitable feeder layers include, by way of example, fibroblasts and epithelial cells, eg, fibroblasts and uterine fibroblasts from chickens, chicken fibroblasts, mouse (eg, mouse or rat) fibroblasts, STO , And S
Includes I-m220 feeder cell line, as well as BRL cells.

[0085] In a preferred embodiment, the feeder cells will comprise mouse embryo fibroblasts. Means for preparing suitable fibroblast layers are described below, which are well known to those skilled in the art.

[0086] The NT units may be cultured in a feeder layer until they reach a suitable size to obtain the cells, and the cells may be used to generate embryonic stem cell-like cells or cell colonies. Preferably, these NT units are at least about 2-400 in size,
More preferably, the cells are cultured until about 4-128, most preferably at least about 500. The cultivation is performed under suitable conditions, ie, about 38.5 ° C., 5% CO ₂ , with the culture medium being typically changed every 2 to 5 days, preferably every 3 days, to optimize growth.

In the case of NT units derived from human cells / enucleated bovine oocytes, enough cells to generate ES cell colonies, typically about 50 cells, have an order of about 50 cells after initiation of oocyte activation. Will be obtained on the 12th. However, this will vary depending on the particular cell used as the nuclear donor, the particular oocyte species, and the culture conditions. Once the desired sufficient number of cells has been obtained, one of ordinary skill in the art can readily ascertain by visual inspection based on the morphology of the cultured NT units.

For human / human nuclear injected embryos, it may be advantageous to use a culture medium known to be useful for maintaining human cells in tissue culture. Examples of culture media suitable for human embryo culture include Jones et al., Human Reprod. 13 (1): 169-177.
(1998), all reported by Irvine, Santa Ana, California.
P1-catalog number 99242 medium available from Scientific, and P-1
Catalog number 99292 media, as well as the media used by Thomson et al., 1998, 1995, supra.

As noted above, the cells used in the present invention will preferably include mammalian somatic cells, most preferably cells from an actively growing (non-quiescent) mammalian cell culture. In a particularly preferred embodiment, the donor cells are genetically modified by the addition, insertion, or replacement of the desired DNA sequence. For example, a donor cell, such as a human, primate, or bovine source, such as a keratinocyte or fibroblast, is transfected or transformed with a DNA construct that provides for expression of a desired gene product, such as a therapeutic peptide. You may. Examples include lymphokines such as IGF-I, IGF-II, interferons, colony stimulating factors, connective tissue polypeptides such as collagen, genetic factors, coagulation factors, enzymes, enzyme inhibitors and the like.

Similarly, as described above, donor cells may be administered prior to nuclear injection to obtain other desirable effects, such as impaired cell lineage development, enhanced embryonic development and / or inhibition of apoptosis. May be modified. Examples of desired modifications are described further below.

One aspect of the invention involves genetically modifying a donor cell, eg, a human cell, so that it is lineage-deficient and therefore cannot produce viable offspring when used for nuclear injection. Will. This is particularly desirable in the case of human nuclear injected embryos, because for ethical reasons it may be an undesirable outcome that viable embryos are produced. This can be done by genetically engineering human cells so that when used for nuclear injection, they cannot differentiate into a particular cell lineage. In particular, the cells, when used as nuclear injection donors, have been genetically modified such that the resulting "embryo" does not contain or is substantially defective in at least one mesoderm, endoderm, or ectoderm tissue. Is also good.

This can be done by knocking out or impairing the expression of one or more mesoderm, endoderm or ectoderm specific genes. Examples include: mesoderm: SRF, MESP-1, HNF-4, β-I integrin, MSD; endoderm: GATA-6, GATA-4 ectoderm: RNA helicase A, Hβ58.

The above list is to be construed as an exhaustive example of known genes involved in mesoderm, endoderm and ectoderm development. The generation of mesodermal, endodermal, and ectodermal defective cells has been previously reported in the literature. For example, Arsenian et al
., EMBO J. 17 (2): 6289-6299 (1998); Saga, Y., Mech. Dev. 75 (
1-2): 53-66 (1998); Holdener et al., Development, Volume 120 (5): 1355-134
6 (1994); Chen et al., Genes Dev. 8 (20): 2466-2477 (1994); Rohwede
l et al., Dev. Biol. 201 (2): 167-189 (1998) (mesoderm): Morrisey et al.
12 (22): 3579-3590 (1998); Soudais et al., Developme.
nt, 121 (11): 3877-3888 (1995) (endoderm); Lee et al., Proc. Natl. Ac
ad. Sci. USA, 95 (23): 13709-13713 (1998) and Radice et al., Develo.
pment, 111 (3): 801-811 (1991) (ectoderm).

In general, the desired somatic cells, eg, human keratinocytes, epithelial cells or fibroblasts, can be “knocked out” such that one or more genes specific for a particular cell lineage are “knocked out”. And / or genetically engineering such that expression of such genes is significantly impaired. This may be done by known methods, for example by homologous recombination. Preferred gene systems for effecting the `` knockout '' of the desired gene are disclosed in Capecchi et al., U.S. Patent Nos. 5,631,153 and 5,464,764, which provide targeted alterations of DNA sequences in the desired mammalian genome. It reports a positive-negative selection (PNS) vector that enables it. Such genetic modification results in cells that cannot be differentiated into a particular cell lineage even when used as a nuclear injection donor.

The use of the genetically modified cells will produce lineage-deficient nuclear injected embryos, ie, embryos that do not develop at least one of functional mesoderm, endoderm, or ectoderm. Thereby, the resulting embryo, even if implanted, for example, in the human uterus, will not give rise to viable offspring. However, ES cells obtained by such nuclear injection are:
It would be useful in that they still yield one or two remaining non-disturbed lineage cells. For example, ectoderm-deficient human nuclear injected embryos will still yield mesodermal and endoderm-derived differentiated cells. Ectodermal deficient cells can be generated by deletion and / or damage of one or both RNA helicase A or Hβ58 genes.

These lineage-deficient donor cells may be genetically modified to express another desired DNA sequence.

Thus, the genetically modified donor cells, when seeded, will produce lineage-deficient blastocysts that differentiate into at most two embryonic germ layers.

[0098] Alternatively, the donor cell may be modified such that it is "lethal." this is,
It can be performed by expressing an antisense or ribozyme telomerase gene. This can be done by known genetic methods that provide for expression of antisense DNA or ribozymes, or by gene knockout. These "lethal" cells will not be able to differentiate into viable progeny when used for nuclear injection.

Another preferred embodiment of the present invention is the production of nuclear injected embryos that grow more efficiently in tissue culture. This is advantageous in that it should reduce the time and fusion required to produce ES cells and / or progeny (when blastocysts are implanted into female surrogate mice). This is desirable because it has been recognized that blastocysts and ES cells obtained by nuclear injection may have impaired developmental potential. While these problems can often be mitigated by changes in tissue culture conditions, another solution is to enhance embryo development by increasing the expression of genes involved in embryo development.

For example, a Ped-type gene product that is a member of the MHC class I family
It has been reported to have significant significance for embryo development. More specifically, in the pre-implantation mouse embryo, the Q7 and Q9 genes have been reported to be associated with a "fast-growing" phenotype. Thus, it is expected that introduction of DNA that provides for expression of these and related genes, or the corresponding DNA of that human or other mammal, into donor cells will result in more rapidly growing nuclear injected embryos. This is particularly desirable in the case of hybrid nuclear injected embryos that are less efficient in tissue culture compared to nuclear injected embryos resulting from fusion of cells or nuclei of the same species.

In particular, a DNA construct comprising the Q7 and / or Q9 genes is introduced into donor somatic cells prior to nuclear injection. For example, a strong constitutive mammalian promoter operably linked to the Q7 and / or Q9 genes, an IRES, one or more suitable selectable markers, such as neomycin ADA, DHFR, and a polyA sequence, such as a bGH polyA sequence. Expression constructs can be constructed. Similarly, it may be advantageous to further enhance Q7 and Q9 gene expression by including an insulating sequence. These genes are expected to be expressed early in blastocyst development because of their high conservation in different species, for example, cows, goats, pigs, dogs and humans. Similarly, it is expected that donor cells can be engineered to affect other genes that enhance embryo development. Thus, these genetically modified donor cells should produce blastocysts and preimplantation embryos more efficiently.

[0102] Yet another aspect of the invention involves the construction of donor cells that are resistant to apoptosis, ie, programmed cell death. Cell death-related genes have been reported in the literature to be present in preimplantation embryos (Adams et al., Scienc
e 281 (5381); 1322-1326 (1998)). Genes reported to induce apoptosis include, for example, Bad, Bok, BH3, Bik, Hrk, BNIP3, Bim _L , Bad, Bid
, And EGL-1. In contrast, genes reported to protect cells from programmed cell death include, by way of example, Bcl-XL, Bcl-w, Mcl-1, A1, Nr
-13, BHRF-1, LMW5-HL, ORF16, Ks-Bel-2, E1B-19K, and CED-9.

Thus, donor cells in which the gene that induces apoptosis is “knocked out” or in which the expression of the gene that protects the cell from apoptosis is enhanced or switched on during embryonic development, Can be built.

For example, this can be done by introducing such a protective gene, eg, a DNA construct that provides for regulated expression of Bcl-2 or a related gene during embryo development. Thereby, the gene can be "switched on" by culturing the embryo under specific growth conditions. Alternatively, it can be linked to a constitutive promoter.

More specifically, regulatable or constitutive promoters, such as PGK, S
A DNA construct comprising the Bcl-2 gene operably linked to V40, CMV, ubiquitin, or β-actin, IRES, a suitable selectable marker, and a polyA sequence is constructed to produce a desired donor mammalian cell, e.g., human keratinocytes. Alternatively, it can be introduced into fibroblasts.

[0106] These donor cells used to generate nuclear-injected embryos should be resistant to apoptosis and thereby differentiate more efficiently in tissue culture. Thereby, the speed and / or number of suitable pre-implantation embryos produced by nuclear injection can be increased.

Another means of achieving the same result is to impair the expression of one or more genes that induce apoptosis. This is by knockout
Alternatively, it is performed by using an antisense or ribozyme against a gene that is expressed early in embryonic development and induces apoptosis. An example is shown above. Alternatively or alternatively, donor cells may be constructed that contain both modifications, ie, damage to the apoptosis-inducing gene and enhanced expression of the gene that interferes with or prevents apoptosis. The construction and selection of genes that affect apoptosis, and cell lines that express such genes, are described in Craig, B.
and Thompson et al. in US Pat. No. 5,646,008. This patent is incorporated herein by reference.

One means of enhancing efficiency is to select cells at a particular cell cycle stage as donor cells. This has been reported to have a significant effect on nuclear injection efficiency. (Barnes et al., Mol. Reprod. Devel. 36 (1): 33-41 (1993)).
Different methods for selecting cells at specific cell cycle stages have been reported, including serum depleted Campbell et al., Nature, 380: 64-66 (1996); Wilmut et al.,
Nature, 385: 810-813 (1997), and Chemical Synchronization Urbani et al., Exp. Cell
Res. 219 (1): 159-168 (1995). For example, a specific cyclin DNA can be operably linked to a regulatory sequence with a detection marker, such as green fluorescent protein (GFP), and then linked to a cyclin disruption box, selectively insulating to enhance cyclin and marker protein expression. Sequences may be combined. Thereby, cells of the desired cell cycle are easily macroscopically detected and selected for use as nuclear injection donors. An example is the cyclin D1 gene for selecting cells in G1. However, any cyclin gene should be suitable for use in the claimed invention (eg, King et al., Mol. Biol. Cell, 7 (9): 1343-1357).
(1996)).

However, there is a need for a less invasive, more efficient method of producing cells of the desired cell cycle stage. This can be done by genetically modifying the donor cells so that they express a specific cyclin under conditions in which they can be detected. Thereby, cells of a particular cell cycle can be easily distinguished from cells of another cell cycle.

[0110] Cyclin is a protein that is expressed only at certain stages of the cell cycle. They include cyclins D1, D2 and D3 in G1, cyclins in G2 / M
Includes B1 and B2, and cyclins E, A and H in S phase. These proteins are easily translated and destroyed in the cytosol. This "transient" expression of such proteins is due, in part, to the presence of "disruption boxes", which are proteins that function as tags that direct rapid destruction of these proteins through the ubiquitin pathway. A short amino acid sequence that is part. (Adams et al., Science, 2
81 (5321): 1322-1326 (1998)).

In the present invention, donor cells expressing one or more such cyclin genes are constructed under conditions that are easily detectable, preferably, for example, conditions that can be visualized by using a fluorescent label. For example, a specific cyclin DNA can be operably linked to a regulatory sequence, along with a detection marker, eg, green fluorescent protein (GFP), and then linked to a cyclin disruption box, selectively insulating to enhance cyclin and marker protein expression. Sequences may be combined. Thereby, cells of the desired cell cycle are easily macroscopically detected and selected for use as nuclear injection donors. An example is the cyclin D1 gene for selecting cells in G1. However, any cyclin gene should be suitable for use in the claimed invention (eg, King et al., Mol. Biol. Cell, 7 (9): 1343-1357 (
1996)).

[0112] Yet a further aspect of the invention is a method of enhancing nuclear injection efficiency, particularly in a hybrid nuclear injection process. We believe that when inserting or fusing different species of enucleated oocytes, nuclei or cells of one species can give rise to nuclear-injected embryos that give rise to blastocysts that can give rise to ES cell lines. The efficiency of the new process proved to be extremely low.
Thus, many fusions typically need to be performed to give rise to blastocysts, and culturing the cells can give rise to ES cells and ES cell lines.

Yet another means of enhancing the development of nuclear-injected embryos in vitro is to optimize culture conditions. One way to achieve this result would be to culture NT embryos in conditions that prevent apoptosis. For this aspect of the invention, it has been found that proteases such as caspases can cause oocyte death by apoptosis similar to other cell types (Jurisicosva et al., Mol. Reprod. Devel. 5).
1 (3): 243-253 (1998)).

[0114] Blastocyst development may be enhanced by including one or more caspase inhibitors in the culture medium used to inject the nuclei and culture the pre-implantation embryo. is expected. Such inhibitors include, by way of example, caspase-4 inhibitor I, caspase-3 inhibitor I, caspase-6 inhibitor II, caspase-9 inhibitor II, and caspase-1 inhibitor I. The amount is an amount effective to inhibit apoptosis, for example, 0.00001 to 5.0% of the medium weight, more preferably 0.01% to 1.0% of the medium weight. Thus, the efficiency of nuclear injection may be increased by enhancing subsequent blastocyst and embryo development in tissue culture using the methods described above.

After the desired size of NT units is obtained, the cells are mechanically removed from the area and used to produce embryonic or stem cell-like cells and cell lines. This is preferably
Obtaining a clump of cells containing NT units, typically comprising at least 50 cells, washing such cells, and seeding the cells on a feeder layer, such as irradiated fibroblasts. Will be Typically, the cells used to obtain stem cell-like cells or cell colonies are obtained from the innermost part of a cultured NT unit, which is preferably at least 50 cells in size. However, smaller or larger cell numbers of NT units may be used along with cells from other parts of the NT units to obtain ES cell-like cells and cell colonies.

Prolonged exposure of donor cell DNA to oocyte cytosol can promote the dedifferentiation process. In this regard, recloning can be performed by taking blastomeres from reconstructed embryos and fusing them with new enucleated oocytes. Alternatively, the donor cells are fused with the enucleated oocytes and 4-6
After a period of time, the chromosome may be removed without activation and fused with a younger oocyte. Activation will then occur.

Cells are maintained on the feeder layer in a suitable growth medium, eg, αMEM supplemented with 10% FCS and 0.1 mM β-mercaptoethanol (Sigma) and L-glutamine. The growth medium may be optionally used to optimize growth, for example, 2-3
Replace every day.

As a result of this culture process, an embryo or stem cell-like cell or cell line is formed.
In the case of NT embryos derived from human cells / bovine oocytes, colonies are observed by 2 days after culture in αMEM medium. However, this time can vary depending on the particular nuclear donor cell, the particular oocyte and the culture conditions. One skilled in the art can vary the culture conditions, if desired, to optimize the growth of a particular embryo or hepatocyte-like cell.

[0119] The resulting embryonic or stem cell-like cells and cell colonies typically have a similar appearance to embryonic or stem cell-like cells of the species used as the nuclear cell donor, rather than the donor oocyte species. Would. For example, in the case of embryo or hepatocyte-like cells obtained by injecting human nuclear donor cells into enucleated bovine oocytes, the cells may be bovine
The morphology is more similar to mouse embryonic stem cells than to ES-like cells.

In more detail, the individual cells of the human ES cell colony are not well-defined and the periphery of the colony is refractive and smooth in appearance. In addition, cell colonies have longer cell doubling times, about twice that of mouse ES cells. Similarly, unlike bovine and porcine derived ES cells, colonies do not have an epithelial-like appearance.

As noted above, primate stem cells are SSEA-1 (-), SSEA-4 (+), TRA-1-60 (+), TRA-1
-81 (+), and alkaline phosphatase (+) are reported by Thomson et al., US Pat. No. 5,843,780. Human and primate ES cells produced according to the present invention will show similar or identical marker expression.

Alternatively, the fact that such cells, indeed humans or primates, are embryonic stem cells will be confirmed on the basis that they can give rise to all of the mesoderm, ectoderm and endoderm tissues. This can be demonstrated, for example, by culturing ES cells produced according to the present invention under suitable conditions, such as disclosed in Thomsen U.S. Pat.No. 5,843,780, which is incorporated herein by reference. There will be.

The resulting embryo or stem cell-like cells and cell lines, preferably human embryo or stem cell-like cells and cell lines, have a number of therapeutic and diagnostic applications. Most particularly, such embryonic or stem cell-like cells may be used for cell transplantation therapy. Human embryos or stem cell-like cells have indications in treating a number of disease conditions.

In this regard, it is known that mouse embryonic stem cells (ES cells) can differentiate into almost any type of cell, for example hematopoietic stem cells. Thus, human embryos or stem cell-like cells generated according to the present invention should have similar potency. The embryo or stem cell-like cells of the invention will be induced to differentiate to obtain the desired cell type according to known methods. For example, by culturing test human embryos or stem cell-like cells in a differentiation medium and conditions that provide for cell differentiation, hematopoietic stem cells, muscle cells, cardiomyocytes, hepatocytes, chondrocytes, epithelial cells, urine Differentiating cells may be induced. The media and methods by which embryonic stem cell differentiation takes place are known in the art, along with suitable culture conditions.

For example, Palacios et al., Proc. Natl. Acad. Sci. USA 92: 7530-7537 (199
5) Incubate such cell clumps first in a suspension culture medium lacking retinoic acid, then in the same medium containing retinoic acid, and then transfer the cell clumps to a substrate that provides cell adhesion Teaching the production of hematopoietic stem cells from embryonic cell lines by subjecting the stem cells to induction techniques, including:

Furthermore, Pedersen, J., Reprod. Fertil. Dev. 6: 543-552 (1994) describes a variety of differentiating cell types including hematopoietic cells, muscle cells, cardiomyocytes, and especially neurons. This is a review citing a number of papers that disclose methods of differentiating embryonic stem cells in vitro.

In addition, Bain et al., Dev. Biol. 168: 342-357 (1995) teaches the in vitro differentiation of embryonic stem cells that give rise to neural cells with neural properties. These references are examples of reported methods for obtaining differentiated cells from embryonic or stem cell-like cells. The disclosures of these references, particularly those relating to methods of differentiating embryonic stem cells, are hereby incorporated by reference in their entirety.

Thus, using known methods and culture media, one skilled in the art can culture the embryo or stem cell-like cells to obtain the desired differentiated cell type, eg, neurons, muscle cells, hematopoietic cells, and the like. May be. In addition, using inducible Bcl-2 or Bcl-xl,
May be useful for enhancing the in vitro development of specific cell lineages. In vivo, Bcl-2 blocks many, but not all, types of apoptotic cell death that occur during lymphatic and neural development. A thorough discussion of how Bcl-2 expression is used to inhibit apoptosis of the relevant cell lineage following transfection of donor cells is provided in U.S. Patent No. 5,646,008, which is incorporated herein by reference. It has been disclosed.

Any desired differentiated cell type may be obtained using the embryonic or stem cell-like cells of the present invention. Therapeutic use of such differentiated human cells is unparalleled. For example, human hematopoietic stem cells may be used in medical treatments requiring bone marrow transplantation. Such techniques are used to treat many diseases, including late stage cancers such as ovarian cancer and leukemia, as well as diseases that compromise the immune system, such as AIDS. Hematopoietic stem cells include, for example, adult somatic cells of cancer or AIDS patients, such as epithelial cells or lymphocytes,
Fusing enucleated oocytes, e.g., bovine oocytes, obtaining embryonic or stem cell-like cells as described above, and culturing such cells under conditions favorable for differentiation until hematopoietic stem cells are obtained. It can be obtained by culturing. Such hematopoietic stem cells may be used for treating cancer and diseases including AIDS.

Alternatively, adult somatic cells of a patient with a neuropathy can be fused with enucleated animal eggs, such as primate or bovine oocytes, human embryos or stem cell-like cells obtained therefrom, and the like. The cells may be cultured under differentiation conditions that result in a neural cell line. Specific diseases that can be treated by transplanting such human nerve cells include, for example, Parkinson's disease, Alzheimer's disease, ALS, and especially cerebral palsy. In certain cases of Parkinson's disease, transplanted fetal brain neurons have been shown to form appropriate connections with surrounding cells and produce dopamine. As a result, a long-term remission of Parkinson's disease syndrome can be obtained.

To allow specific selection of differentiated cells, the donor cells are transfected with a selectable marker expressed through an inducible promoter, thereby selecting for a particular cell lineage when differentiation is induced or Concentration may be allowed. For example, CD34-neo is used to select hematopoietic cells, Pw-1-neo is used to select muscle cells, Mash-1-neo is used to select sympathetic nerves, and Mal-neo is used to select human CNS neurons in cerebral cortical gray matter. May be used.

A great advantage of the present invention is that it provides an essentially unlimited supply of homologous or syngeneic human cells suitable for transplantation. Thus, an important problem associated with current transplantation methods, namely, rejection of transplanted tissue that may occur due to host-to-graft or graft-to-host rejection, will be avoided. Traditionally, rejection
Prevented or reduced by administering an anti-rejection drug such as cyclosporine. However, such drugs not only have significant side effects, such as immunosuppression, carcinogenic properties, but are also very expensive. The present invention should eliminate, or at least greatly reduce, the need for anti-rejection drugs.

Other diseases and conditions that can be treated by allogeneic cell therapy include, for example, spinal cord injury, multiple sclerosis, muscular dystrophy, diabetes, liver disease, ie, hypercholesterolemia, heart disease, cartilage replacement, burns, feet. Ulcers, gastrointestinal diseases, vascular diseases, renal diseases, urinary tract diseases, and aging-related diseases and conditions.

Similarly, human embryos or stem cell-like cells produced according to the present invention may be used to produce genetically engineered or transgenic human differentiated cells. In essence, this may involve introducing the desired gene or genes, which may be heterologous, or all or one of the endogenous gene or genes of a human embryo or stem cell-like cell produced according to the invention. This will be done by removing parts and by allowing such cells to differentiate into the desired cell type. A preferred method of obtaining such alterations is homologous recombination, because such techniques involve inserting, deleting, or deleting a gene or genes at a specific site or sites in the stem cell-like cell genome. Or it can be used for modification.

The methodology is intended to replace defective genes, such as defective immune system genes, cystic fibrosis genes, or thereby, with growth factors, lymphokines, cytokines,
It can be used to introduce genes that express therapeutically useful proteins such as enzymes. For example, a gene encoding a brain-derived growth factor may be introduced into human embryos or stem cell-like cells, and neural cells and cells that have been differentiated into cells may be transplanted into Parkinson's disease patients to transform neural cells during such diseases. Loss may be delayed.

To date, cell types transfected with the BDNF gene have varied from primary cultured cells to immortalized cell lines, neural or non-neural (myoblasts and fibroblasts) -derived cells. For example, astrocytes were transfected with the BDNF gene using a retroviral vector, and the cells were transplanted into a rat model of Parkinson's disease (Yoshimoto et al., Brain Research, 691: 25-36 (1995)).

This ex vivo treatment was performed 32 days after injection of Parkinson's disease symptoms in rats.
Reduced to 45%. Similarly, introduction of the tyrosine hydroxylase gene into astrocytes gave similar results (Lundberg et al., Develop. Neurol. 139: 39-53 (
1996) and references cited therein).

[0138] However, such ex vivo systems have problems. In particular, currently used retroviral vectors are down-regulated in vivo and transgenes are only transiently expressed (Mulligan, Science, 260: 926-932).
(1993)). Similarly, such tests used primary cultured cells, stellate cells, which have a finite lifespan and slow replication. Such properties have a deleterious effect on the transfection rate and prevent the selection of stably transfected cells. Moreover, it is almost impossible to grow large populations of genetically indexed primary cells used in homologous recombination techniques.

In contrast, the problems associated with retroviral systems should be eliminated by using human embryos or stem cell-like cells. It has already been shown by the present proxy that bovine and porcine embryo cell lines can be transfected and selected for stable integration of heterologous DNA. Such a method is disclosed in U.S. Patent Application Serial No .: filed April 1, 1996, which is hereby incorporated by reference in its entirety.
08 / 626,054. Thus, using such or other known methods, the desired gene is introduced into a test human embryo or stem cell-like cell and the cells are transformed into the desired cell type, such as hematopoietic cells, neural cells, pancreatic cells, cartilage cells. The cells may be differentiated.

Examples of genes that may be introduced into the embryo or stem cell-like cells of the present invention include epidermal growth factor, basic fibroblast growth factor, glial-derived neurotrophic growth factor, insulin-like growth factor (I and II), neurotropin-3, neurotropin-4 / 5, ciliary neurotrophic factor, AFT-1, cytokine genes (interleukin, interferon, colony stimulating factor, tumor necrosis factor (α and β), etc.), treatment Genes encoding enzymes, collagen, human serum albumin and the like are included.

Further, if necessary, one of the negative selection systems currently known in the art can be used to eliminate therapeutic cells from a patient. For example, donor cells transfected with the thymidine kinase (TK) gene lead to the production of embryonic cells containing the TK gene. Differentiation of these cells will isolate those therapeutic cells that also express the TK gene. Such cells will be selectively eliminated from the patient at any time upon administration of ganciclovir. Such a negative selection system is described in US Pat. No. 5,698,446, which is incorporated herein by reference.

The embryonic or stem cell-like cells of the present invention, preferably human cells, may also be used as an in vitro model of differentiation, particularly for examining genes involved in the regulation of early development.

Similarly, cell tissues and organs differentiated using the embryo or stem cell-like cells of the present invention may be used in drug testing.

In addition, the embryo or stem cell-like cells of the present invention may be used as a nuclear donor for producing other embryo or stem cell-like cells and cell colonies.

The following examples are provided to more clearly illustrate the present invention.

Example 1 Materials and Methods Donor Cells for Nuclear Injection Epithelial cells were gently scraped from a consenting adult mouth into a standard glass slide. Cells were washed from the glass slides into Petri dishes containing phosphate buffered saline without Ca or Mg. Cells were pipetted with a small-bore pipette to break up cell clumps into single cell suspensions. The cells were then transferred under oil into microdroplets of TL-HEPES medium containing 10% fetal calf serum (FCS) for nuclear injection into enucleated bovine oocytes.

Nuclear Injection Techniques Basic nuclear injection techniques have been described. Briefly, after maturation of the oocytes in the slaughterhouse in vitro, the cumulus cells were detached from the oocytes and the cells were matured at approximately
Enucleation was performed with a beveled micropipette at 18 hours (hpm). Enucleation is TL-HE
It was confirmed in PES medium plus bisbenzimide (Hoechst 33342, 3 μg / ml, Sigma). Individual donor cells were placed in the perivitelline space of recipient oocytes. Bovine oocyte cytoplasm and donor nuclei (NT units) were fused using electrofusion techniques. One fusion pulse of 90 V for 15 microseconds was applied in NT units. This was done 24 hours after the onset of oocyte maturation (hpm). NT units were placed in CR1aa medium up to 28 hpm.

The techniques used to artificially activate oocytes have been described in the literature.
Activation in NT units was 28 hpm. Briefly, the activation technique is as follows: NT units were exposed to TL-HEPES solution containing 1 mg / ml BSA of ionomycin (5 μM; Calbiochem, La Jolla, Calif.) For 4 minutes. , 30 m
Washed for 5 minutes in TL-HEPES with g / ml BSA. Next, NT units were transferred to small water drops of CR1aa culture medium containing 0.2 mM DMAP (Sigma), and cultured at 38.5 ° C., 5% CO ₂ for 4 to 5 hours. After washing the NT units, 10% FCS and 6 mg / ml BSA in a 4 well plate containing a confluent feeder layer of mouse embryo fibroblasts.
(See below). The NT unit was further cultured at 38.5 ° C. and 5% CO ₂ for 3 days. The culture medium was changed every three days until 12 days after activation. At this point, the NT units have reached the desired cell number, ie, about 50 cells have been mechanically removed from the band and used to generate an embryonic cell line. FIG. 1 shows a photograph in NT units obtained as described above.

(Fibroblast Feeder Layer) Primary cultures of embryonic fibroblasts were obtained from 14-16 day old mouse fetuses. Pre-warmed trypsin ED after aseptically removing the head, liver, heart and digestive tract, and then shredding the embryo
Incubated for 30 minutes at 37 ° C. in a TA solution (0.05% trypsin / 0.02% EDTA; Gibco, Grand Island, NY). The fibroblasts were seeded in tissue culture flasks and 10% fetal calf serum (FCS) (HiClone, Logan, UT), penicillin (100 IU / ml), and streptomycin (50 μl / ml)
) Was added to an α-MEM medium (BioWittucker, Walkersville, MD). Embryonic fibroblasts were irradiated in 35 × 10 Nunc tissue culture dishes (Baxter Scientific, McGraw Park, Ill.) 3-4 days after passage. Irradiated fibroblasts were incubated in a humid atmosphere containing 5% CO ₂ for 37 hours.
The cells were grown and maintained at 0 ° C. The embryo cell line was cultured using a culture plate having a uniform cell monolayer.

(Production of Embryonic Cell Line) The NT unit cells obtained as described above were washed and seeded directly on irradiated feeder fibroblasts. These cells included cells in the inner part of the NT unit. Cells were maintained in a growth medium consisting of αMEM supplemented with 10% FCS and 0.1 mM β-mercaptoethanol (Sigma). The growth medium was changed every 2-3 days.
The first colonies were observed by the second day of culture. When the colonies are expanded, they show a morphology similar to the previously disclosed mouse embryonic stem cells (ES cells). Individual cells within the colony are not clearly demarcated, and the periphery of the colony is refractive and smooth in appearance. Cell colonies appear to have a slower cell doubling time than mouse ES cells. Similarly, unlike bovine and porcine derived ES cells, colonies do not have the epithelial cell appearance as before. 2 to 5 are photographs of ES-like cell colonies obtained as described above.

(Production of Differentiated Human Cells) The obtained human embryo cells were transferred to a differentiation medium and cultured until a differentiated human cell type was obtained.

(Results) Table 1. Human cells as donor nuclei in NT unit production and development

[Table 1]

One NT unit developed into a structure of 16 cells or more was seeded on the fibroblast feeder layer. When this structure was attached to the feeder layer and growth was initiated, a colony having an ES cell-like morphology was formed (see, for example, FIG. 2). Moreover, the 4-16 cell stage structure was not used in attempts to form ES cell colonies, but it has been shown that this stage can produce ES or cell lines for ES (mouse, Eistet
ter et al., Devel. Growth and Differ. 31: 275-282 (1989); cattle, Stice e
t al., 1996). Thus, it is expected that 4-16 cell stage NT units should give rise to embryonic or stem cell-like cells and cell colonies.

Similarly, similar results were obtained for the fusion of an adult human keratinocyte cell line cultured in a medium containing ACM, uridine, glucose, and LIF 1000 IU with enucleated bovine oocytes. 22 out of 50 reconstructed embryos split and one developed into a blastocyst at about day 12. The blastocysts are seeded and the production of ES cell lines is in progress.

Although the invention has been described and illustrated herein with reference to various specific materials, methods and examples, the present invention is not limited to this specific material, combination of materials, and choices for that purpose. It is understood that the invention is not limited to the method described. Numerous modifications of such details will occur, and will be appreciated, by those skilled in the art.

[Brief description of the drawings]

FIG. 1 is a photograph of a nuclear injection (NT) unit produced by injecting human adult cells into enucleated bovine oocytes.

FIG. 2 is a photograph of embryonic stem-like cells obtained from the NT unit as shown in FIG.

FIG. 3 is a photograph of embryonic stem-like cells obtained from the NT unit as shown in FIG.

FIG. 4 is a photograph of embryonic stem-like cells obtained from the NT unit as shown in FIG.

FIG. 5 is a photograph of embryonic stem-like cells obtained from the NT unit as shown in FIG.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 1/16 A61P 13/02 3/10 17/02 9/14 19/00 11/00 19/08 13 / 02 21/00 17/02 25/02 101 19/00 25/14 19/08 25/16 21/00 25/18 25/02 101 25/28 25/14 31/18 25/16 35/00 25 / 18 C12N 5/00 B 25/28 A61K 37/48 31/18 37/02 35/00 37/24 // C12N 15/09 C12N 15/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN , CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG , SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, UZ, VN, YU, ZA, ZW (71) Applicant University of Massachusetts, A Public Institution of Higher Eduquet Ision of the Common Wealth Roble, Massachusetts Settlement, As Representative by It's Amherst Campus Massachusetts, Amherst, United States of America, Goodell Building, Room 512, Office of Vice Chancellor for Research at Amherst, Commercial Bencharders and Interlectual Property (72) James United States Massachusetts, Belchartown, Old Infield 196 (72) Inventor Sibel, Joes United States Massachusetts, Amherst, Bridge Park 166 (72) Inventor Stace, Stephen, El United States Massachusetts, Belchertown, Amherst Road 468 F term (reference) 4B024 AA01 BA80 CA01 DA02 EA04 FA02 FA10 GA09 GA10 GA11 HA17 4B065 AA90X AA93Y AB01 AB0 4 AB06 BA01 BA08 CA24 CA44 CA60 4C084 AA02 AA14 BA08 BA23 CA25 DA11 DB52 DC01 MA66 NA10 NA14 ZA022 ZA162 ZA362 ZA592 ZA752 ZA812 ZA892 ZA942 ZA962 ZB262 ZC352 ZC552 4C087 AA01 AA02 BB63 CA04 Z12A66 ZA92A66 ZC35 ZC55

Claims (50)

[Claims] 1. A method for producing an embryonic cell or stem-like cell comprising the following steps: (i) Inserting into a differentiated human or mammalian cell suitable for the purpose or an animal oocyte denucleated from a cell nucleus Collecting such oocytes from an animal species other than human or mammalian cells under conditions suitable for forming a nuclear injection (NT) unit; (ii) activating the resulting nuclear injection unit (Iii) culturing the activated nuclear injection unit until the two-cell division stage or later; and (iv) producing cells obtained from the cultured NT unit to produce embryonic cells or stem-like cells. Culturing to 2. The method according to claim 1, wherein the cell to be inserted into the enucleated animal oocyte is a human cell. 3. The method according to claim 2, wherein said human cells are adult cells. 4. The method according to claim 2, wherein said human cells are epithelial cells, keratinocytes, lymphocytes or fibroblasts. 5. The method according to claim 2, wherein the oocyte is collected from a mammal. 6. The method according to claim 5, wherein the animal oocyte is collected from an ungulate. 7. The method according to claim 6, wherein said ungulate is selected from the group consisting of cows, sheep, pigs, horses, capine, and buffalo. 8. The method of claim 1, wherein the enucleated oocyte is matured prior to enucleation. 9. The method of claim 1, wherein the fused nuclear injection unit is activated in vitro. 10. The method according to claim 1, wherein the activated nuclear injection unit is cultured on a feeder layer culture. 11. The method according to claim 10, wherein the feeder layer is a fibroblast. 12. The method according to claim 1, wherein the cells in step (iv) from an NT unit having 16 or more cells are cultured on a feeder cell layer. 13. The method according to claim 12, wherein the feeder cell layer is a fibroblast. 14. The method according to claim 13, wherein the fibroblast is a mouse embryonic fibroblast. 15. The method according to claim 1, which induces differentiation of the obtained embryonic cells or stem-like cells. 16. The method according to claim 2, which induces the differentiation of the obtained embryonic cells or stem-like cells. 17. The method of claim 1, wherein the fusion is performed by electrofusion. 18. An embryonic cell or stem-like cell obtained according to the method of claim 1. 19. A human embryonic cell or stem-like cell obtained according to the method of claim 2. 20. A human embryonic cell or stem-like cell obtained according to the method of claim 3. 21. A human embryonic cell or stem-like cell obtained according to the method of claim 4. 22. A human embryonic cell or stem-like cell obtained according to the method of claim 6. 23. A human embryonic cell or stem-like cell obtained according to the method of claim 7. 24. A differentiated human cell obtained by the method of claim 16. 25. The method according to claim 25, wherein the cells are selected from the group consisting of nerve cells, hematopoietic cells, pancreatic cells, muscle cells, chondrocytes, urinary cells, hepatocytes, spleen cells, germ cells, skin cells, butterfly cells, and gastric cells. 25. The differentiated human cell according to claim 24, which is selected. 26. A method for treating a patient in need of cell transplantation therapy, wherein human cells differentiated according to claim 24 are administered. 27. The cell transplantation therapy according to claim 26, wherein Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, spinal cord defect or injury, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, cartilage defect or 27. The method according to claim 26, wherein the method is used for treating a disease or medical condition selected from the group consisting of injury, burn, leg ulcer, vascular disease, urinary tract disease, AIDS and cancer. 28. The differentiated human cell is a hematopoietic cell or a neural cell.
26. The method according to 26. 29. The method of claim 26, wherein the treatment is for the treatment of Parkinson's disease and the differentiated cells are neurons. 30. The method of claim 26, wherein the therapy is for the treatment of cancer and the differentiated cells are hematopoietic cells. 31. The differentiated human cell according to claim 24, which contains and expresses the inserted gene. 32. The method according to claim 1, wherein a gene suitable for the purpose is inserted, deleted or modified in the embryonic cell or stem-like cell. 33. The method of claim 32, wherein the gene of interest encodes a therapeutic enzyme, growth factor, or cytokine. 34. The method according to claim 32, wherein said embryonic cells or stem-like cells are human embryonic cells or stem-like cells. 35. The method according to claim 32, wherein the gene suitable for the purpose is removed, modified or deleted by homologous recombination. 36. The method of claim 1, wherein the donor cells are genetically modified to inhibit development of at least one of the endoderm, ectoderm and mesoderm. 37. The method of claim 1, wherein the donor cells are genetically modified to increase differentiation efficiency. 38. The method according to claim 36, wherein the cultured nuclear injection unit is cultured in a medium containing at least one capsase inhibitor. 39. The method according to claim 1, wherein the donor cell expresses a detection marker indicating the expression of a specific cyclin. 40. SRF, MESP-1, HNF-4, beta-1, integrin, MSD, GATA-6
37. The method of claim 36, wherein the donor cells have been modified to alter expression of a gene selected from the group consisting of: GATA-4, RNA helicase A, and Hbeta 58. 41. The method of claim 37, wherein said donor cells have been genetically modified to introduce DNA expressing the Q7 and / or Q9 genes. 42. The method of claim 41, wherein said gene is operably linked to a regulatable promoter. 43. The method of claim 1, wherein the donor cells have been genetically modified to inhibit apoptosis. 44. Bad, Bok, BH3, Bik, Blk, Hrk, BNIP3, Gim L, Bid, EGL-1,
Bcl-XL, Bcl-w, Mcl-1, Al, Nr-13, BHRF-1, LMW5-HL, ORF16, Ks-Bcl-2, E1B-1
44. The method of claim 43, wherein apoptosis is reduced by altering expression of one or more genes selected from the group consisting of 9K and CED-9. 45. The method of claim 44, wherein at least one of said genes is operably linked to an inducible promoter. 46. A mammalian somatic cell which expresses a DNA encoding a detectable marker, the expression of which is associated with a particular cyclin. 47. The cell according to claim 46, wherein the cyclin is selected from the group consisting of cyclins D1, D2, D3, B1, B2, E, A, and H. 48. The cell of claim 46, wherein said detectable marker is a fluorescent polypeptide. 49. The cell of claim 48, wherein said mammalian cell is selected from the group consisting of human, primate, rodent, ungulate, dog, and cat cells. 50. The cell of claim 48, wherein said cell is a human, bovine, or primate cell.
The cell according to 1.