JP2002505100A - Embryonic or stem cell-like cell lines generated by interspecies nuclear transfer - Google Patents

Abstract

  (57) [Summary] An improved method of nuclear transfer is provided that includes transferring the differentiated donor cell nucleus to an enucleated oocyte of a different species from the donor cell. The obtained nuclear transfer unit is useful for producing isogenic embryonic stem cells, particularly human isogenic embryonic stem cells. These embryonic or stem cell-like cells are useful for generating desired differentiated cells and for introducing, removing or modifying the desired gene, eg, by homologous recombination, at a particular genomic site in such cells. These cells may carry heterologous genes and are particularly useful for cell transplantation therapy and in vitro studies of cell differentiation. Also provided is a method of improving nuclear transfer efficiency by genetically modifying donor cells to inhibit apoptosis, selecting cells in a specific cell cycle, and / or enhancing embryonic growth and development. Is done.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention generally produces embryonic or stem cell-like cells by transplanting cell nuclei from an animal or human into enucleated oocytes of an animal of a different species than the donor nucleus. About things. The invention more particularly relates to animal oocytes enucleated from nuclei of primate or human cells, such as primate or ungulate oocytes, and in preferred embodiments bovine enucleated oocytes The invention relates to making primate or human embryonic or stem cell-like cells by transplanting into cells. The present invention further relates to the use of the resulting embryonic or stem cell-like cells, preferably primate or human embryonic or stem cell-like cells, in therapeutic, diagnostic applications, as well as in differentiated cells used in therapeutic or diagnostic applications. The invention relates to the use for the production and for the production of transgenic embryonic or transgenic differentiated cells, cell lines, tissues and organs. In addition, the embryonic or stem cell-like cells obtained according to the invention may themselves be used as nuclear donors in nuclear transfer or nuclear transfer procedures for producing chimeras or clones, preferably transgenic clones or chimeric animals.

BACKGROUND OF THE INVENTION Methods for deriving embryonic stem (ES) cells from early unimplanted mouse embryos in vitro are well known (eg, Evans et al., Nature, 29: 154-156 (1981). ); Martin, Proc. Natl.
Acad. Sci., USA, 78: 7634-7638 (1981)). ES cells can be passaged in an undifferentiated state, provided that the fibroblast feeder layer is present (Evans et al., Supra) and that a source of differentiation inhibition is present (Smith et al., Dev. Biol. ., 121: 1-9 (1
987)). It has been reported that ES cells have various applications. For example,
It has been reported that ES cells can be used as an in vitro model of differentiation, particularly for studying genes involved in controlling early development. Mouse ES cells can generate germline chimeras when introduced into unimplanted mouse embryos, thus demonstrating their pluripotency (Bradely et al., Nature, 309: 255- 256 (1984)).

[0003] Because of the ability to deliver their genome to the next generation, ES cells have potential utility for livestock germline engineering using ES cells with or without the desired genetic modification. It has nature. In addition, in the case of livestock animals, for example, ungulates, nuclei from embryos, such as unimplanted livestock embryos, support the development of enucleated oocytes to term (Smit
H et al., Biol. Reprod., 40: 1027-1035 (1989); and Keefer et al., Biol. Reprod.
, 50: 935-939 (1994)). This is symmetric with the nucleus of the mouse embryo. Mouse embryo nuclei reportedly do not support the development of enucleated cells beyond the eight cell stage after transplantation (Cheong et al., Biol. Reprod., 48: 958 (1993)). Therefore, for livestock animals
ES cells are highly desirable because they may provide a potential source of totipotent, genetically engineered or unengineered donor nuclei for nuclear transfer methods.

[0004] Several research groups have reported the isolation of a cell line referred to as a pluripotent embryonic cell line. For example, Notarianni et al., J. Reprod. Fert. Suppl., 43: 255-260 (1
991) report that a stable multipotent cell line was established from porcine and ovine blastocysts. This cell line was obtained from sheep blastocysts immunosurgically (imm
unosurgically) show similar morphological and growth characteristics of isolated inner cell mass with cells in primary culture (Id.). Also, Notarianni et al., J. Reprod. Fert. Suppl., 41:
51-56 (1990) discloses the maintenance and differentiation in culture of pluripotent embryonic cell line candidates from porcine blastocysts. Further, Gerfen et al., Anim. Biotech, 6 (1): 1-14 (1995
) Discloses the isolation of embryonic cell lines from porcine blastocysts. These cells are stably maintained in the mouse embryonic fibroblast feeder layer without using a conditioning medium. These cells are reportedly differentiated into several differentiated cell types in culture (Gerfen et al., Supra).

[0005] In addition, Saito et al., Roux's Arch. Dev. Biol., 201: 134-141 (1992) report a cultured bovine embryonic stem cell-like cell line, which has been passaged three times. But was lost after the fourth passage. Furthermore, Handyside et al., Roux's Arch Dev.
Biol., 196: 185-190 (1987) disclose culturing the inner cell mass of immunosurgically isolated sheep embryos under conditions that allow the isolation of mouse ES cells from mouse ICM. I have. Handyside et al., (1987) (ibid.) Describe that under such conditions sheep ICM adheres and spreads, generating both ES and endoderm-like cell areas, but after long-term culture, Only cells are reported to be prominent (Id.). Recently, Cherny et al., Theriogenology, 41: 175 (1994) reported bovine primordial germ cells, referred to as pluripotent, derived from a cell line maintained in long-term culture. These cells formed ES-like colonies after approximately 7 days in culture, stained positively for alkaline phosphatase, exhibited the ability to form embryoid bodies, and spontaneously differentiated into at least two cell types. These cells also reportedly have transcription factors OCT4, OCT6
And the pattern of the homeobox gene, which expresses HES1 mRNA and is thought to be expressed exclusively by ES cells.

In recent years, Campbell et al., Nature, 380: 64-68 (1996) have reported that cultured scutellum (ED) from ovine day 9 embryos cultured in mice under conditions that facilitated the isolation of ES cell lines. ) We reported the production of live sheep by nuclear transfer of cells. The authors, based on their own results,
It was concluded that ED cells from sheep day 9 embryos were totipotent by nuclear transfer and that totipotency was maintained during culture. Van Stekelenburg-Hamers et al., Mol. Reprod. Dev., 40: 444-454 (1995) reported the isolation and characterization of cells, termed permanent cell lines, from the inner cell mass of bovine blastocysts. The authors isolated the ICM from bovine day 8 or day 9 blastocysts and cultured them under various conditions to determine which feeder cells and media were the most effective in supporting bovine ICM adhesion and subsequent growth. Determined to be effective. Based on their own results,
Adherence and subsequent expansion of cultured ICM cells (instead of bovine uterine epithelial cells)
It was concluded that it was enhanced by the use of feeder cells (mouse fibroblasts) and by the use of charcoal-stripped serum to supplement the medium (rather than normal serum). However, Van Stekelenburg et al. Reported that their cells more closely resemble epithelial cells than pluripotent ICM cells (Id.).

Further, Smith et al., WO 94/24274, published October 27, 1994, Evans et al., WO 90/034
32, published April 5, 1990, and Wheelre et al., WO 94/26889, published November 24, 1994, isolate and select animal stem cells that may be used to obtain transgenic animals. And proliferation. Also, Evans et al., WO 90/03432, published April 5, 1990, reported the induction of embryonic stem cells from pigs and cattle, termed pluripotent, which claim to be useful in the generation of transgenic animals. . In addition, Wheeler et al., WO 94/26884, published November 24, 1994, disclosed embryonic stem cells that are claimed to be useful in generating chimeric and transgenic ungulates. Thus, based on the above, numerous groups have attempted to create ES cell lines, for example, due to their potential applicability in the generation of cloned or transgenic embryos and nuclear transfer.

[0008] The use of ungulate ICM cells for nuclear transfer has also been reported. For example, Colloas et al.
Mol. Reprod. Dev., 38: 264-267 (1994) discloses nuclear transfer of bovine ICM into enucleated mature oocytes by microinjection of lysed donor cells. This document disclosed that embryos were cultured in vitro for 7 days to give rise to 15 blastocysts, which, when implanted in bovine recipients, gave birth to four and two were born. Also, Keefer et al., Biol. Reprod., 50: 935-939 (1994) discloses the use of bovine ICM cells as donor nuclei for blastocyst generation in nuclear transfer procedures. The blastocysts gave rise to several offspring when transplanted into bovine recipients. Furthermore, Sims et al.
Proc. Natl. Acad. Sci., USA, 90: 6143-6147 (1993)
Calf production by nuclear transfer of nuclei from ICM Saito into enucleated mature oocytes was disclosed. The production of live sheep by nuclear transfer of cultured scutellum cells has also been reported (Campb
ell et al., Nature, 380: 64-68 (1996)). In addition, the use of bovine pluripotent embryonic cells in nuclear transfer and generation of chimeric bovine fetuses has been reported (Stice et al., Biol. Repr.
od., 54: 100-110 (1996)); Collas et al., Mol. reprod. Dev., 38: 264-267 (1994).

There have also been previous attempts to produce interspecies NT units (Wolfe et al., Theriogeno
logy, 33: 350 (1990)). Specifically, bovine embryonic cells were fused with bison oocytes to create several interspecies NT units that appeared to have an inner cell mass.
However, embryonic cells rather than adult cells were used as donor nuclei in the nuclear transfer procedure. Dogma says that embryonic cells are more easily reprogrammed than adult cells. This dates back to early NT studies on frogs (Diberardin
o Differentiation, 17: 17-30 (1980)). This study also involves animals that are phylogenetically very similar (bovine nuclei and bison egg cells). On the contrary,
Previously, if a wider variety of species had been fused during NT (bovine nuclei into hamster oocytes), no inner cell mass structure was obtained. Furthermore, it has never previously been reported that the inner cell mass from NT units could be used to form ES cell-like colonies capable of growth. Collas et al. (Id.) Also teach the use of granulosa cells (adult somatic cells) for the generation of bovine nuclear transfer embryos. However, unlike the present invention, these experiments did not involve interspecies nuclear transfer. Also, unlike the present invention, no ES-like cell colonies were obtained.

Quite recently, given to James A. Thomson on December 1, 1998, Wiscons
U.S. Patent No. 5,843,780, assigned to the Alumni Research Foundation, intends to disclose the following purified preparations of primate embryonic stem cells. The cells are capable of (i) growing in vitro for more than one year; (ii) having all the chromosomes characteristic of primate species and a karyotype that does not change significantly over long-term culture. (Iii) maintain the ability to differentiate during culture into endoderm, mesoderm and ectoderm tissue-derived products; and (iv) do not differentiate when cultured on fibroblast feeder layers.
These cells were reported negative for the SSEA-1 marker, positive for the SEA-3 marker,
SEA-4 marker positive, expresses alkaline phosphatase activity, is pluripotent, contains the presence of all chromosomes characteristic of primate species, and has a karyotype in which no chromosomes have been altered. I have. Furthermore, these cells are TRA-1-60 and TRA-1-81 marker positive. When these cells are injected into SCID mice, endoderm, mesoderm,
It is said to differentiate into ectoderm cells. Also referred to as human or primate blastocyst-derived embryonic stem cell lines are Thomson et al., Science 282: 1145-1147 and Proc.
tl. Sci., USA 92: 7844-7848 (1995).

[0011] Thus, Thomson discloses cells referred to as non-human primate and human embryonic or stem cell-like cells and methods for their production. However, there is still a great need for methods of producing human embryonic or stem cell-like cells that are autologous to the intended transplant recipient and have been given significant therapeutic and diagnostic capabilities. In this regard, numerous human diseases have been identified that may be treated by cell transplantation. For example, Parkinson's disease is caused by degeneration of dopaminergic neurons in the substantia nigra. The standard treatment for Parkinson's disease involves the administration of L-DOPA, which temporarily ameliorates dopamine deficiency but causes serious side effects and does not eventually reverse the progression of the disease. Different approaches to the treatment of Parkinson's disease have the potential for broad application in the treatment of many brain diseases and central nervous system injuries, but involve transplanting fetal or neonatal cells or tissues into the adult brain . Fetal nerves from various regions of the brain can be taken into the adult brain. Such transplantation has been shown to reduce experimentally induced behavioral deficits, including complex cognitive functions, in experimental animals. Initial results from human clinical trials are also promising. However, the supply of human fetal cells or tissues by abortion is very limited. Furthermore, obtaining cells or tissues from aborted fetuses is very controversial.

[0012] Currently, there is no available method for producing "fetal-like cells" from a patient. Furthermore, allografts are not readily available, and both allografts and xenografts are subject to graft rejection. Further, in some cases, it may be beneficial to make genetic modifications to the cells or tissues before transplantation. However, many cells or tissues for which such modification is desired do not divide very much in culture, and most genetic transformations require rapidly dividing cells. Thus, there is a clear need in the art for transplantation and the supply of human embryonic or stem cell-like undifferentiated cells for use in cell and gene therapy.

OBJECTS OF THE INVENTION It is an object of the present invention to provide a new and improved method for producing embryonic or stem cell-like cells. A more specific object of the present invention is a new and improved method for producing embryonic or stem cell-like cells, comprising transplanting the nucleus of a mammalian or human cell into an enucleated oocyte of a different species It is to provide. Another object of the present invention is, in particular, to transfer the nucleus of a non-human primate or human cell into an enucleated animal or human oocyte, such as an ungulate, human or primate enucleated oocyte To provide a new and improved method for producing non-human primates or human embryonic or stem cell-like cells. Another object of the present invention is to provide a non-human primate or human cell, such as a line comprising engrafting a non-human primate or human oocyte enucleated from the nucleus of a human adult cell.
A novel method for producing defective non-human primate or human embryonic or stem cell-like cells, wherein such cells are modified to a specific cell lineage, for example, by manipulating the expression of an antisense or ribozyme telomerase gene. Has been genetically engineered to be unable to differentiate into cells, or has been modified such that the cells are "dead"
It is an object of the present invention to provide a method which does not produce viable offspring.

It is yet another object of the present invention to genetically engineer donor somatic cells used for nuclear transfer to enhance embryonic development, such as MHC I family genes, particularly Q7 and / or Q9. By expressing such a Ped gene, the efficiency of nuclear transfer is increased, and in particular, the development of unimplanted embryos produced by nuclear transfer is promoted. Yet another object of the present invention is to enhance the production of nuclear transfer embryos by IVP, and more particularly, to make the donor cells used for nuclear transfer resistant to apoptosis, such as a gene that inhibits apoptosis. Genetically modifying it to be apoptotic by, for example, expressing Bcl-2 or a Bcl-2 family member and / or expressing an antisense ribozyme specific for a gene that induces apoptosis during early embryonic development Is to enhance the production of nuclear transfer embryos. It is yet another object of the present invention to genetically modify a donor cell to express a DNA construct encoding a particular cyclin linked to a detectable marker, such as a visualizable (eg, fluorescent tag) marker protein. The goal is to improve the efficiency of nuclear transfer by improving the selection of donor cells at a particular cell cycle stage, eg, the G1 phase.

[0015] It is also an object of the present invention to culture embryos produced in vitro in the presence of one or more protease inhibitors, preferably in the presence of one or more caspase inhibitors, thereby inhibiting apoptosis. Thereby enhancing embryonic development. It is another object of the present invention to provide embryonic or stem cell-like cells produced by transplanting animal or human cells into enucleated oocytes of different species. The purpose of the present invention is in particular, animal cells enucleated primate or human cells, for example,
To provide primate or human embryonic or stem cell-like cell cells by transplanting nuclei into human, primate or ungulate enucleated oocytes. Another object of the invention is to use such embryonic or stem cell-like cells for therapy or diagnosis. The purpose of the present invention is in particular to use such primate or human embryonic or stem cell-like cells for the treatment or diagnosis of any disease for which cell, tissue or organ transplantation is therapeutically or diagnostically beneficial. That is. Another object of the invention is to use the embryonic or stem cell-like cells produced according to the invention for the production of differentiated cells, tissues or organs.

It is an object of the invention, inter alia, to use the primate or human embryonic or stem cell-like cells produced according to the invention for the production of differentiated human cells, tissues or organs. Another object of the present invention is to provide an embryonic or stem cell-like cell produced according to the present invention,
Generation of genetically engineered embryonic or stem cell-like cells that can be used to create genetically engineered or transgenic differentiated human cells, tissues or organs, e.g., cells that can be used for gene therapy It is used for fabrication. Another object of the invention is in particular the use of embryonic or stem cell-like cells produced according to the invention in vitro, for example for studying cell differentiation, and for assay purposes, for example for drug research. . Another object of the invention is to provide an improved method of transplantation therapy, comprising the use of an isogenic or syngenic cell, tissue or organ made from embryonic or stem cell-like cells made according to the invention. That is. Such treatments include, for example, Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, spinal cord injury, multiple sclerosis, muscular dystrophy, diabetes, liver disease, heart disease, cartilage replacement, burn, vascular disease, It includes the treatment of diseases and injuries including urinary tract disease, and among other diseases, immunodeficiency, bone marrow transplantation, cancer.

Another object of the invention is to use the transgenic or genetically engineered embryonic or stem cell-like cells produced according to the invention for gene therapy, in particular for the treatment and / or prevention of the above mentioned diseases and injuries. It is to be. Another object of the invention is an embryonic or stem cell-like cell produced according to the invention,
Alternatively, the use of the transgenic or genetically engineered embryonic or stem cell-like cells produced according to the present invention as a nuclear donor for nuclear transfer. Yet another object of the present invention is to use the genetically engineered ES cells generated according to the present invention for the production of transgenic animals, such as non-human primates, rodents, ungulates and the like. That is. Such transgenic animals can be used, for example, to create animal models for the study of human disease, or can be used to produce desired polypeptides, such as for therapeutic or nutritional medicine. it can. The foregoing and other objects, advantages and features of the invention will become apparent hereinafter. The nature of the invention will be more clearly understood by reference to the following detailed description of preferred embodiments of the invention and the appended claims.

Description of the Invention The present invention provides a novel method for producing embryonic or stem cell-like cells, and more specifically, non-human primate or human embryonic or stem cell-like cells. In the present application, nuclear transfer, nuclear transfer or NT are used interchangeably. As discussed above, nuclear transfer of embryonic or stem cell-like cells, ie, actual isolation by nuclear transfer, has never been reported before. Rather, the previously reported isolation of ES-like cells was from fertilized embryos. Furthermore, successful nuclear transfer involving cells or DNA of genetically different species, more specifically adult cells or DNA of one species (eg human) and oocytes of another unrelated species There have been no reports of successful nuclear transfer. Rather, there have been reports of embryos produced by the fusion of cells of closely related species (eg, bovine and goat and bovine and bison), but such embryos did not produce ES cells (Wolfe et al. al, Theriogenology,
33 (1): 350 (1990)). Furthermore, no method has been reported for producing primate or human ES cells derived from non-fetal tissues. On the contrary, the limited human fetal cells and tissues currently available must be obtained from accidentally aborted tissues and aborted fetuses. Furthermore, prior to the present invention, no one had obtained embryonic or stem cell-like cells by interspecies nuclear transfer.

Quite unexpectedly, we have found that human embryonic or stem cell-like cells and cell colonies can be used to replace the nuclei of human cells (eg, adult differentiated cells) with enucleated oocytes of animals. (Transplantation was used to generate nuclear transfer (NT) units, and the cells of the nuclear transfer unit were cultured in human embryonic or stem cell-like cells and cell colonies. Yields). This result is very surprising. This includes cells that give rise to embryonic or stem cell-like cells and colonies when the differentiated donor cells or nuclei are introduced into enucleated oocytes of genetically different species and cultured under appropriate conditions Effective interspecies nuclear transfer involving the generation of a nuclear transfer unit, for example, the first to perform a transfer of cell nuclei obtained from differentiated animal or human cells (eg, adult cells) to enucleated eggs of a different animal species That's why.

[0020] Preferably, the NT units used to generate ES-like cells are cultured to a size of at least 2 to 400 cells, preferably 4 to 128 cells, most preferably at least about 50 cells. In the present invention, embryonic or stem cell-like cells refer to cells made according to the present invention. In this application, we refer to stem cell-like cells rather than stem cells for the typical manner in which they are made, ie, because they are made by interspecies nuclear transfer. These cells are expected to have a differentiation ability similar to normal stem cells,
There will be some differences that are not important due to the manner in which they were made.
For example, these stem cell-like cells have the mitochondria of the oocyte used for nuclear transfer and therefore do not behave identically to normal embryonic stem cells.

The present invention provides that the transfer of nuclei of adult cells, especially human epithelial cells obtained from the oral cavity of a human donor, results in the formation of nuclear transfer units when transferred to bovine oocytes, The cells were made based on the finding that they give rise to human stem cell-like or embryonic cells and human embryonic or stem cell-like cell colonies in culture. Recently, the results showed that adult keratinocytes were transplanted into bovine enucleated oocytes and blastocysts and
Reproduced by the ability to create ES cell lines.

Based on this, we have found that bovine oocytes and human oocytes, and more generally mammals, undergo a sufficiently similar or conserved maturation process during embryonic development, It has been hypothesized that bovine oocytes can function as effective substitutes or substitutes for human oocytes. Obviously, oocytes in general will likely contain proteinaceous or nucleic acid factors that induce embryo development under appropriate conditions, and the same or very similar functions in different species. Important for these factors
RNA and / or telomerase may be included.

Based on the fact that human cell nuclei can be effectively transplanted into bovine oocytes, human cells can be transferred to oocytes of other unrelated species (eg, other animals as well as other ungulates). It is reasonable to expect it to be portable. In particular, other ungulates (eg pigs,
Oocytes from sheep, horses, goats, etc.) should be appropriate. Furthermore, oocytes from other sources may also be suitable (eg, oocytes from other primates, amphibians, rodents, rabbits, guinea pigs, etc.). Furthermore, human cells or cell nuclei could be transplanted into human oocytes using a similar method, and human ES cells could be produced using the obtained undifferentiated germ cells.

Thus, in its broadest embodiment, the invention relates to the implantation of an animal or human cell nucleus or animal or human cell into an enucleated oocyte of a different animal species than the donor nucleus by implantation or fusion. And making NT units that can be used to obtain embryonic or stem cell-like cells and / or cell cultures. For example, the present invention provides that ungulate or ungulate cells can be implanted or implanted into enucleated oocytes of another species (eg, another ungulate or non-ungulate) by implantation or fusion of these cells and And / or combining the nuclei to produce NT units, which may comprise multicellular NT units (preferably including at least about 2 to 400 cells, more preferably including 4 to 128 cells, and most preferably including at least 50 cells). Culturing under appropriate conditions to obtain. By culturing using cells of such NT units, embryonic or stem cell-like cells or cell colonies can be produced.

However, preferred embodiments of the present invention are directed to human, primate, or non-primate oocytes (eg, ungulate oocytes) from human human cell nuclei or enucleated human cells. Embodiments include making non-human primate or human embryonic or stem cell-like cells by transplanting into bovine enucleated oocytes.

In general, embryonic or stem cell-like cells are produced by a nuclear transfer process that includes the following steps: (i) obtaining a human or animal cell desired to be used as a source of donor nucleus (donor nucleus); (Ii) obtaining oocytes from a suitable source (eg, from mammals and most preferably from primates or ungulates, eg, cattle); (iii) removing the oocytes (Iv) transferring the human or animal cells or nuclei to enucleated oocytes of a different animal species than the donor cells or nuclei, for example by fusion or injection; (v) the resulting NT product or NT Culturing the unit to produce a multicellular structure; and (vi) culturing cells from the embryo to obtain embryonic or stem cell-like cells and stem cell-like colonies.

Nuclear transfer or nuclear transfer techniques are known in the literature and are described in many of the references cited in the prior art. See, for example, in particular: Campbe
ll et al, Theriogenology, 43: 181 (1995); Collas et al, Mol. Report Dev.,
38: 264-267 (1994); Keefer et al, Biol. Reprod., 50: 935-939 (1994); Sims et.
al, Proc. Natl. Acad. Sci. USA, 90: 6143-6147 (1993); WO94 / 26884; WO94 / 24
274; and WO 90/03432, which are incorporated herein by reference. Furthermore, U.S. Pat. Nos. 4,944,384 and 5,074,420 disclose a method for nuclear transfer of cattle. See also the following references: Cibelli et al, Sci
ence, 280: 1256-1258 (1998).

[0028] 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, by way of example, epithelial cells, nerve cells, epidermal cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), other immune cells Erythrocytes, macrophages, monocytes, monocytes, fibroblasts, cardiomyocytes and other muscle cells. In addition, human cells used for nuclear transfer are available from various organs, such as skin, lung, pancreas, liver, stomach, intestine, heart, genitalia, bladder, kidney, ureter and other urinary organs. These are only examples of suitable donor cells. Suitable donor cells, ie, cells useful in the present invention, can be obtained from any cell or organ of the body. These include all somatic or germ cells. Preferably, the donor cell or donor nucleus comprises actively dividing cells (ie, non-resting cells) because it has been reported to enhance the cloning effect. More preferably, such donor cells will be in the G1 phase of the cells.

The obtained undifferentiated germinal cells are described in the literature (Thomson et al, Science 282: 1145-1147 (1
Natl. Acad. Sci. USA, 92: 7544-7848 (1995), which are incorporated herein by reference in their culture method as reported in Thomson et al. Proc. Natl. Can be used to obtain stem cell lines. In the examples below, cells used as donors for nuclear transfer were epithelial cells from the oral cavity of human donors and human adult keratinocytes. However, as discussed above, the disclosed method is applicable to other cells or nuclei in humans.
In addition, cell nuclei can be obtained from both human somatic and germ cells.

In addition, donor cells can be arrested at mitosis prior to nuclear transfer using appropriate techniques known in the art. Methods for arresting the cell cycle at various stages are reviewed in detail in US Pat. No. 5,262,409, which is incorporated herein by reference. In particular, although cycloheximide has been reported to have an inhibitory effect on mitosis (Bowen & Wilson, J. Heredity 45: 3-9 (1995)), it has also been reported that mature bovine follicular It can be used to improve the activation of mother cells (Yang et al, Biol. Reprod. 42 (Suppl.
1): 117 (1992)).

Activation of the zygote gene is associated with hyperacetylation of histone H4. Trichostatin-A, like other compounds, has been shown to inhibit histone deacetylase in a reversible manner (Adenot et al, "Differential H4 acetylation of pa
ternal and maternal chromatin precedes DNA replication and Differential
transcriptional activity in pronuclei of 1-cell mouse embryos ". Developm
ent 124 (22): 4615-4625 (Nov. 1997); Yoshida et al, "Trichostatin A and tra
poxin: novel chemical probes for the role of histone acetylation in chro
matessa structure and function ". Bioessays 17 (5): 423-430 (May, 1995)).

For example, butyrate is also believed to cause histone hyperacetylation by inhibiting histone deacetylase. In general, butyrate appears to modify gene expression, and in almost all cases, the addition of butyrate to cultured cells appears to stop cell growth. The use of butyrate in this context is described in US Pat. No. 5,681,718, which is incorporated herein by reference. Thus, the donor cells can be exposed to trichostatin-A or another suitable deacetylase inhibitor prior to fusion. Also, such compounds can be added to the culture solution prior to genome activation.

In addition, demethylation of DNA is thought to be a requirement for proper access of transcription factors to DNA regulatory sequences. Global demethylation from the 8-cell stage to the blastocyst stage of the embryo before transfer has been described previously (Stein et al, Mol. Reprod. & Dev. 47 (4): 121-429). It was also reported that 5-azacytidine can be used to reduce cellular DNA methylation levels (Jaenisch et al, (1997)). This may lead to increased access of transcription factors to DNA regulatory sequences. Thus, donor cells may be exposed to 5-azacytidine (5-Aza) prior to fusion. Also 5-Az
a may be added to the culture solution from the 8-cell stage to the blastocyst stage. Alternatively, other known methods can be used to perform DNA demethylation.

Oocytes used for nuclear transfer can be obtained from animals, including mammals and amphibians. Suitable mammalian sources of oocytes include cows, sheep, pigs, horses, rabbits, goats, guinea pigs, mice, hamsters, rats, primates, humans, and the like. In a preferred embodiment, the oocyte is obtained from a primate or ungulate (eg, a cow). Methods for isolating oocytes are well known in the art. Essentially, this involves isolating oocytes from the ovaries or genital tract of a mammal (eg, a cow) or amphibian. A readily available source of bovine oocytes is slaughterhouse material.

For successful use of techniques such as genetic manipulation, nuclear transfer and cloning, oocytes are generally fertilized before using these cells as recipient cells for nuclear transfer and by sperm cells. It must be matured in vitro before it can be allowed to develop into an embryo. This process generally involves collecting immature (early I) oocytes from the ovaries of an animal (eg, bovine ovaries obtained at the slaughterhouse) and, prior to fertilization or enucleation, transforming these oocytes into metaphases. It requires maturation in a ripening broth until II is reached, which generally occurs about 18-24 hours after aspiration for bovine oocytes. For the purposes of the present invention, this period is known as the "aging period".
As used herein in the calculation of this period, "aspiration" refers to aspiration of immature oocytes from a follicle.

In addition, metaphase II oocytes matured in vivo have been successfully used in nuclear transfer techniques. Essentially, from non-superovulated or superovulated or heifers, 35-48 hours after the onset of estrus or the same time after injection of human chorionic gonadotropin (hCG) or similar hormones After passage, mature metaphase II oocytes were harvested surgically.

The stage of oocyte maturation upon enucleation or transfer of nuclei was reported to be important for the success of the NT method (see, eg, Prather et al, Differentiati
on, 48: 1-9 (1991)). Generally, metaphase II oocytes have been used as recipient oocytes in successful mammalian embryo cloning techniques to date. Because the oocyte is processing the fertilized sperm, it may be considered "activated" enough at this time to process the introduced nucleus. In domestic animals (especially cattle), the duration of oocyte activation generally ranges from about 16 to 52 hours, preferably from about 28 to 42 hours after inhalation.

For example, immature oocytes are available from HEPES-buffered hamster embryo culture (HECM) in the literature (Ses
Washed as described in hagine et al, Biol. Reprod. 40: 544-606 (1989)) and then allowed to stand under a drop of mature culture at 39 ° C under a layer of lighter paraffin or silicon. The mature culture is prepared with a suitable gonadotropin (eg, luteinizing hormone (
LH) and follicle stimulating hormone (FSH)) and estradiol.
Consist of 50 microliters of 199% tissue culture medium (TCM) containing fetal bovine serum.

After a predetermined maturation time (typically about 10 to 40 hours, preferably about 16-
18 hours), the oocytes were enucleated. Prior to enucleation, oocytes are preferably removed and placed in HECM containing 1 milligram / milliliter hyaluronidase before removing cumulus cells. Removal of cumulus cells can be achieved by repeated aspiration with a very fine inside diameter pipette or by brief vortexing. The exposed oocytes were subsequently screened for polar bodies and selected for metaphase II
Oocytes (determined by the presence of polar bodies) are subsequently used for nuclear transfer. The enucleation is as follows.

Enucleation can be performed by known methods, for example, the methods described in US Pat. No. 4,994,384, which is incorporated herein by reference. For example, metaphase II oocytes can be placed in HECM (optionally containing 7.5 micrograms / milliliter of cytokerasin B) for immediate enucleation, or in a suitable culture (eg, CR1aa + estrous phase). The enucleation can be carried out after standing in 10% of the serum of a cow, but preferably not after 24 hours, more preferably after 16-18 hours.

Enucleation can be accomplished by microsurgery using a micropipette to remove the polar body and nearby cytoplasm. Subsequently, the oocytes are screened to identify those that have been successfully enucleated. This screening can be performed by staining the oocytes with 1 microgram / milliliter of 33342 Hoechst dye in HECM, followed by observing the oocytes under ultraviolet light for less than 10 seconds. The oocytes that have been successfully enucleated are subsequently placed in the appropriate culture medium. In the present invention, the recipient oocyte is preferably about 1 hour after the onset of in vitro maturation.
0 to about 40 hours, more preferably about 16 hours to about 24 hours after the onset of in vitro maturation.
The enucleation is carried out for a period of time, most preferably about 16 to 18 hours after the onset of in vitro maturation.

A single animal or human cell, typically heterologous to an enucleated oocyte, or a nucleus derived therefrom, is subsequently transferred to the perivitelline space of an enucleated oocyte used to make NT units. . NT units are made using animal or human cells or nuclei and enucleated oocytes according to methods known in the art. For example, cells can be fused by electrofusion. Electrofusion can be achieved by providing sufficient electrical pulses to cause transient plasma membrane disruption.
This disruption of the plasma membrane is very brief since the membrane is repaired quickly. Basically, when disruption is induced in two adjacent membranes, upon reformation, the lipid bilayer mixes and small channels open between the two cells. Due to the thermodynamic instability of such a small opening, the opening is enlarged until the two cells become one. For further discussion of this method, see US Pat. No. 4,997,384 (Prather et al.).
al), which is incorporated herein by reference. A variety of electrofusion media can be used, including, for example, sucrose, mannitol, sorbitol, and phosphate buffered saline. Fusion can also be achieved using Sendai virus as a fusogenic agent (Graham, Wister Inot. Sym.
p. Monogr. 9:19 (1969)).

Furthermore, in some cases (eg, in the case of small donor nuclei), it is preferable to inject the nuclei directly into the oocyte rather than using electroporation fusion. Such techniques are described in the literature (Collas & Barnes, Mol. Reprod. Dev. 38: 264-267 (1994
This document is hereby incorporated by reference). Preferably, human or animal cells and oocytes are electrofused in a 500 μm compartment by applying a 90-120 V electric pulse for about 15 μsec about 24 hours after the onset of oocyte maturation. After fusion, until activation (eg, as shown below)
The obtained fusion NT unit is allowed to stand in an appropriate culture solution. Typically, activation is performed shortly thereafter, typically after less than 24 hours, preferably after about 4-9 hours.

The NT unit can be activated in a known manner. Such methods include, for example, culturing the NT unit at a sub-physiological temperature (essentially by applying a cold (actually cold) shock to the NT unit). This can be most conveniently performed by culturing the NT unit at room temperature. Room temperature is cold compared to the physiological temperature conditions to which the embryo is normally exposed. Alternatively, activation can be achieved by the application of known activating substances. For example,
Oocyte entry by sperm at fertilization activates the pre-fusion oocytes, resulting in a number of vitally active pregnancies, resulting in a number of genetically identical calves after nuclear transfer.
In addition, treatments such as electrical and chemical shock, or cycloheximide treatment, can also be used to activate NT embryos after fusion. Suitable oocyte activation methods are the subject of US Pat. No. 5,496,720 (Susko-Parrish et al), which is hereby incorporated by reference.

For example, oocyte activation can be achieved simultaneously or sequentially by performing the following steps: (i) increasing the level of divalent cations in the oocyte, and (ii) increasing the oocyte Reduces the phosphorylation of cellular proteins. This is generally achieved by introducing a divalent cation, such as magnesium, strontium, barium or calcium, into the cytoplasm of the oocyte, for example in the form of an ionophore. Other methods of increasing divalent cation levels include the use of electric shock, ethanol treatment, and treatment with a caged chelator. Phosphorylation can be reduced by known methods, such as the addition of kinase inhibitors such as serine-threonine kinase inhibitors such as 6-dimethyl-amino-purine, staurosporine, 2-aminopurine, and sphingosine. . Alternatively, phosphorylation of cellular proteins can be suppressed by introducing phosphatases (eg, phosphatase 2A and phosphatase 2B) into the oocyte.

Activated NT units can be cultured in suitable in vitro culture media until the generation of embryonic or stem cell-like cells and cell colonies. Suitable culture media for the culture and maturation of embryonic or stem cell-like cells are well known in the art. Examples of known cultures, which can be used to culture and maintain bovine embryonic or stem cell-like cells, include ham F
-10 + 10% fetal calf serum (FCS), tissue culture solution 199 (TCM-199)
+ 10% fetal calf serum, Tyrode-albumin-lactate-pyruvate (T
ALP), Dulbecco's phosphate buffered saline (PBS), Eagle and Witten cultures. One of the most common cultures used for oocyte collection and maturation
One is TCM-199 and 1-20% serum supplement (including fetal calf serum, neonatal serum, estrus cow serum, lamb serum or castrated bull serum). A preferred maintenance culture is TCM-199 containing Earle's salts, 10% fetal bovine serum, 0.2M
Contains M Ma pyruvate and 50 μg / ml gentamicin sulfate. Any of the above can include co-cultures containing a variety of cell types (eg, granulosa cells, fallopian tube cells, BRL cells and uterine cells and STO cells).

In particular, human epithelial cells of the endometrium secrete leukemia inhibitory factor (LIF) before and during implantation. Therefore, the addition of LIF to the culture medium will
It may be important for enhancing in vitro development. The use of LIF in embryonic or stem cell-like cell culture is described in US Pat. No. 5,712,156, which is incorporated herein by reference. Another maintenance culture is described in US Pat. No. 5,096,822 (Rosenkrans, Jr. et al), which is incorporated herein by reference. This embryo culture solution (C
R1) contains the nutrients required to maintain the embryo. CR1 contains hemicalcium L-lactate in an amount ranging from 1.0 mM to 10 mM, preferably 1.0 mM to 5.0 mM. Hemicalcium L-lactate is L-lactate that has incorporated therein a hemicalcium salt. Further, suitable culture media for maintaining human embryonic cell cultures are as described in the literature (Thomson et al, Science 282: 1145-1147 (1998) and Proc.
Natl. Acad. Sci. USA, 92: 7844-7848 (1995)).

Thereafter, the culture NT unit is preferably washed, followed by a suitable culture, such as CRIaa culture, Ham's F-10, tissue culture-199 (TCM-199),
Tyrode-albumin-lactate-pyruvate (TALP) Dulbecco's phosphate buffered saline (PBS), Eagle or Witten culture (preferably about 1
(Including 0% FCS). Such a culture can be performed in a well plate containing a suitable confluent feeder layer. Exemplary suitable feeder layers include fibroblasts and epithelial cells, eg, fibroblasts and uterine epithelial cells from ungulates, chicken fibroblasts, murine (eg, mouse or rat)
, STO and SI-m220 feeder cell lines, and BRL cells.

In a preferred embodiment, the feeder cells will comprise mouse embryo fibroblasts. Means for preparing a suitable fibroblast feeder layer are described in the Examples below, but are well within the skill of those in the art. NT units are cultured on the feeder layer until they reach the appropriate size to obtain cells that can be used to create embryonic stem cell-like cells or cell colonies. Preferably, these NT units comprise at least about 2 to 400 cells,
More preferably it will be cultured until it reaches a size of about 4 to 128 cells, most preferably at least 50 cells. Culture is performed under appropriate conditions (ie, about 38.5 ° C).
And 5% CO ₂ ), typically with a media change every about 2-5 days, preferably about every 3 days, to optimize growth.

In the case of NT units from human cells / enucleated bovine oocytes, sufficient cells (typically on the order of about 50 cells) to produce ES cell colonies are Will be obtained in about 12 days from the start of activation. However, this will vary according to the individual cells used as nuclear donors, individual oocyte species, and culture conditions. One skilled in the art can easily confirm when a desired sufficient number of cells can be obtained based on the form of the cultured NT unit. In the case of human / human nuclear transfer embryos, it may be advantageous to use a culture that is known to be useful in maintaining human tissue culture cells. Examples of cultures suitable for human embryos include cultures reported in the literature (Jones et al, Human Reprod. 13 (1): 169-177 (1
998)), P1-catalog number # 99242 culture and P1-catalog number # 9
9292 culture solution (both Irvine Scientific)
(Available from Santa Ana, Calif.), And Thomson et al. ((199
8) and (1995) supra).

As discussed above, the cells used in the present invention preferably include mammalian somatic cells, most preferably cells from actively dividing (non-quiescent) mammalian cell cultures. In a particularly preferred embodiment, the donor cells will be genetically modified by the addition, deletion or substitution of the desired DNA sequence. For example, donor cells (eg, keratinocytes or fibroblasts (eg, human, primate or bovine)
)) Is transfected or transformed with a DNA construct that provides for expression of the desired gene product, eg, a therapeutic peptide. Examples of therapeutic polypeptides include lymphokines (eg, IGF-I, IGF-II), interferons, colony stimulating factors, connective tissue polypeptides (eg, collagen), genetic factors, coagulation factors, enzymes, enzyme inhibitors, and the like. As discussed further above, the donor cells are modified prior to nuclear transfer to achieve a desired effect (eg, development of an impaired cell lineage, enhanced embryonic development, and / or suppression of apoptosis). Can be. Examples of desired modifications are discussed further below.

One of the features of the present invention involves genetic modification of donor cells (eg, human cells) that are defective lines and thus cannot produce viable offspring when used for nuclear transfer. This is desirable where the generation of viable embryos is an undesirable result for ethical reasons, especially in the case of human nuclear transfer embryos. This can be accomplished by genetically manipulating human cells so that they cannot differentiate into a particular cell lineage when used for nuclear transfer. In particular, when used as a nuclear transfer donor, the cells can be genetically modified such that the resulting "embryo" does not contain or is substantially devoid of at least one of mesoderm, endoderm or ectoderm tissue. It is envisioned that this can be achieved by knocking out or damaging one or more mesoderm, endoderm or ectoderm specific genes. Examples of such genes 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 representative of known genes involved in mesoderm, endoderm or ectoderm development and is intended to be a non-exclusive list. The development of mesodermal, endodermal or ectodermal deficient cells and embryos has been previously reported in the literature. See, for example, Arsenian et al, EMBO J. 17 (2): 6289-6299 (1998); Y. Saga, Mech. De.
v. 75 (1-2): 53-66 (1998); Holdener et al, Development, 120: (5): 1355-1346 (1
994); Chen et al, Genes Dev. 8 (20): 2466-2477 (1994); Rohedel et al, Dev.
Biol. 201 (2): 167-189 (1998) (mesoderm); Morrisey et al, Genes Dev. 12 (22):
3579-3590 (1998); Soudais et al, Development 121 (11): 3877-3888 (1995) (endoderm); and Lee et al, Proc. Natl. Acad. Sci. USA, 95 (23): 13709- 13713 (19
98); Radice et al, Development 111 (3): 801-811 (1991) (ectoderm).

In general, the desired somatic cells (eg, human keratinocytes, epithelial cells, or fibroblasts) are “knocked out” by one or more genes specific to a particular cell lineage, and And / or will be genetically engineered so that expression of such genes is significantly impaired. This can be achieved by known methods (eg, homologous recombination). A preferred genetic system for effecting "knock-out" of a desired gene is described in U.S. Pat.
No. 4. The patent discloses a positive-negative selection (PNS) vector that allows targeted directed modification of DNA sequences in the desired mammalian genome. Such genetic modifications will result in cells that cannot be differentiated into a particular cell lineage when used as a nuclear transfer donor.

The genetically modified cells are used to produce a lineage-defective nuclear transfer embryo, ie, an embryo that does not develop at least one of functional mesoderm, endoderm or ectoderm. . Thereby, the resulting embryo will not give rise to viable offspring even if implanted, for example, in the human uterus. However, ES cells obtained from such nuclear transfer are useful in that they produce one or two lines of residual undamaged cells. For example, ectoderm-deficient human nuclear transfer embryos will give rise to mesodermal and ectoderm-derived differentiated cells. Ectodermal deficient cells can be created by deletion and / or damage of one or both of the RNA helicase A or Hβ58 genes.

[0056] These defective lineage donor cells can also be genetically modified to express another desired DNA. Thus, the genetically modified donor cell gives rise to a defective lineage blastocyst, which when transplanted will differentiate at most two germ layers. Alternatively, the donor cells can be modified to be "lethal". This can be achieved by expressing the antisense or ribozyme telomerase gene.
This can be done by known genetic methods that provide for expression of antisense DNA or ribozymes, or by knocking out genes. These “lethals”
The cells will not be able to differentiate into viable progeny when used for nuclear transfer.

Another preferred embodiment of the present invention is the generation of a nuclear transfer embryo that grows more efficiently in tissue culture. This is advantageous in that it reduces the time and fusion required to produce ES cells and / or progeny (when blastocysts are transferred to surrogate mothers). This is also desirable as it has been observed that blastocysts and ES cells generated by nuclear transfer may be impaired in development. While these problems are often mitigated by altering tissue culture conditions, another solution is to enhance embryo development by enhancing expression of genes required for embryo development.

For example, Ped-type gene products, which belong to the MHC-I family, have been reported to be crucial for embryonic development. More specifically, in pre-implantation mouse embryos, the Q7 and Q9 genes were reported to be required for the "rapid growth" phenotype. Thus, introduction of DNA that provides for expression of these and related genes, or their human or other mammalian counterparts, into donor cells will result in more rapidly growing nuclear transfer embryos. It is expected. This is especially desirable in the case of interspecies nuclear transfer embryos that exhibit less efficient development in tissue culture than nuclear transfer embryos resulting from fusion of cells or nuclei of the same species.

In particular, DNA constructs containing the Q7 and / or Q8 genes will be introduced into donor somatic cells prior to nuclear transfer. For example, the Q7 and / or Q8 genes, IRES, one or two
Expression constructs can be constructed that include a strong constitutive mammalian promoter operably linked to one or more selectable markers (eg, neomycin, ADA, DHFR) and a poly-A sequence (eg, a bGH polyA sequence). It would also be beneficial to further enhance Q7 and Q9 gene expression by adding shielding sequences (insulates). Because these genes are highly conserved in different species (eg, cows, goats, pigs, dogs, cats, and humans), they are expected to be expressed early in blastocyst development. Furthermore,
It is expected that the donor cells can be manipulated to affect other genes that enhance embryonic development. Thus, these genetically modified donor cells should produce blastocysts and pre-implantation stage embryos more efficiently.

Another aspect of the invention involves the construction of a donor cell that is resistant to apoptosis (ie, programmed cell death). Genes associated with cell death have been reported in the literature to be present in preimplantation stage embryos (Adams et al, Science 281).
(5381): 1322-1326 (1998)). Genes reported to induce apoptosis include:
Bad, Bok, BH3, Bik, Hrk, BNIP3, BiM L, Bad, include Bid and BGL-1. Conversely, genes reported to protect cells from programmed cell death include, for example, Bc
L-XL, Bcl-w, Mcl-1, A1, Nr-13, BHRF-1, LMW5-HL, ORF16, Ks-Bel-2, E1B-19K and CED
-9 is included.

Thus, donor cells can be constructed in which the gene that induces apoptosis has been “knocked out” or in which the expression of genes that protect cells from apoptosis is enhanced or initiated during embryonic development. For example, this can be accomplished by introducing a DNA construct that provides for regulated expression of such a protective gene (eg, Bc1-2) or a related gene during embryonic development. Thus, this gene can be "triggered" by culturing the embryo under specific growth conditions. Alternatively, the gene may be linked to a constitutive promoter.

More specifically, the Bcl-2 gene linked to a regulatable or constitutive promoter (eg PGK, SV40, CMV, ubiquitin or β-actin, IRES), a suitable selectable marker and a poly-A sequence Can be constructed and introduced into a desired donor mammalian cell (eg, a human keratinocyte or fibroblast). When used to generate nuclear transfer embryos, these donor cells should be resistant to apoptosis and thereby differentiate more efficiently in tissue culture. Thus, the rate and / or number of suitable pre-implantation embryos produced by nuclear transfer can be increased.

Another means of achieving the same result is to attenuate the expression of one or more genes that induce apoptosis. This may be achieved by knockout or by using antisense or ribozymes against genes that are expressed in embryonic development and induce apoptosis early in embryonic development. The example is the same as above. Alternatively, donor cells may be constructed to contain both modifications (ie, enhanced apoptosis-inducing gene damage and expression of genes that slow or prevent apoptosis). Construction and selection of genes that affect apoptosis, and cell lines that express such genes are described in US Pat.
No. 6008 (Craig B. Thompson et al., Assigned to the University of Michigan), which is hereby incorporated by reference.

One way to enhance efficiency is to select cells in a particular cell cycle as donor cells. This was reported to have a significant effect on nuclear import efficiency (Barmes et al, Mol. Reprod. Devel. 36 (1): 33-41 (1993)). Various methods for sorting cells in a particular cell cycle phase have been described, including serum deprivation (Campbell et al, Nature, 380: 64-66 (1966); Wilmut et al, Nature, 385: 810
-813 (1977)) and entrainment by chemicals (Urbani et al, Exp. Cell Res. 219 (1
): 159-168 (1995)).

For example, a particular cyclin DNA can be operably linked to a regulatory sequence along with a detectable marker (eg, green fluorescent protein (GFP)), the cyclin break box, and optionally the cyclin and marker. This is followed by an insulation sequence that enhances protein expression. Thereby, cells having a desired cell cycle to be used as a nuclear transfer donor can be easily detected and sorted visually. An example is the cyclin D1 gene for sorting cells in the G1 phase. However, any cyclin gene should be suitable for use in the present invention (see, eg, King et al, Mol. Biol. Cell 7 (9)).
: 1343-1357 (1996)). However, there is a need for a less invasive and more efficient method for producing cells at the desired cell cycle stage. This is expected to be possible with donor cells that have been genetically modified to express a particular cyclin under detectable conditions. This allows cells in a particular cell cycle to be easily distinguished from other cell cycles.

Cyclin is a protein that is expressed only at certain stages of the cell cycle.
These include cyclins D1, D2 and D3 in G1, cyclins B1 and B2 in G2 / M,
And S-phase cyclins E, A and H. These proteins are easily translated and destroyed by cytogolcytosol. This "transient" expression of such proteins is due, in part, to the presence of a "disruption box". The disruption box is a short amino acid sequence that is part of a protein that functions as a tag to induce immediate destruction of these proteins by the ugiquitin pathway (Adams et al, Science 281 (5321): 1322-1326 (1998 )).

In the present invention, donor cells that express one or more such cyclin genes under conditions that are readily detectable, preferably in a visible manner (eg, by use of a fluorescent label) Will be built. For example, a particular cyclin DNA can be operably linked to a regulatory sequence, preferably with a detectable marker (eg, green fluorescent protein (GEP)), a cyclin disruption box, and optionally a cyclin and / or marker. This is followed by a shielding sequence that enhances protein expression. Thereby, cells of the desired cell cycle to be used as nuclear transfer donors can be easily detected and sorted visually. An example is G1
This is a cyclin D1 gene that can be used to select cells in the anaphase. However, any cyclin gene should be suitable for use in the present invention (see, eg, King et al, Mol. Biol. Cell 7 (9): 1343-1357 (1996)). .

Still another aspect of the present invention is a method for improving the efficiency of nuclear transfer, preferably the efficiency of an interspecies nuclear transfer method. We can produce a nuclear transfer embryo that forms a blastocyst when a nucleus or cell of one species is inserted or fused into an enucleated oocyte of a different species, the embryo having an ES Although proven to give rise to cell lines, the efficiency of such methods is very low. Thus, many fusions typically need to be streamlined to form blastocysts. Here, blastocyst cells are sometimes cultured to give rise to ES cells and ES cell lines. Furthermore, another method for enhancing the development of nuclear transfer embryos in vitro is by optimizing culture conditions. One way to achieve this result is to culture NT embryos under conditions that inhibit apoptosis. For this embodiment of the invention, it has been found that proteases such as caspases can cause oocyte death by apoptosis similar to other cell types (Jurisi
cosava et al., Mol. Reprod. Devel., 51 (3): 243-253 (1998)).

The development of blastocysts includes one or more caspase inhibitors in the medium used for nuclear transfer, maintenance of the blastocysts, or culture of the embryo at the unimplanted stage. It is expected that the 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. . Their amount will be an amount effective to inhibit apoptosis, for example from 0.00001 to 5.0% by weight relative to the medium; more preferably from 0.01% to 1.0% by weight relative to the medium. Thus, the methods described above could be used to increase the efficiency of nuclear transfer by enhancing blastocyst and embryo development in subsequent tissue culture. Once the desired size of NT units has been obtained, the cells are mechanically removed from the zone and then used to generate embryonic or hepatocyte cells and cell lines. This is preferably an aggregate containing NT units, which typically contains at least about 50 cells, but the aggregate is taken, the cells are washed, and the cells are plated on a feeder layer. For example, provided by plating on irradiated fibroblasts. However, lower or higher cell numbers of NT units and cells from other parts of the NT units may also be used to obtain ES-like cells and cell colonies.

Longer exposure of the donor cell DNA to the oocyte cytoplasm may facilitate the differentiation process. In this regard, recloning can be achieved by removing the split cells from the reconstituted embryos and by honoring them with new enucleated oocytes. Alternatively, 4-6 hours after fusing the donor cells with the enucleated cells, without activation,
Chromosomes can be removed and fused with younger oocytes. Activation will then occur. Cells are maintained in a 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 is frequently added as needed to optimize growth, for example,
Replace every 2-3 days. This culture process results in the formation of embryonic or stem cell-like cells or cell lines. In the case of NT embryos derived from human cells / bovine oocytes, colonies are observed by about 2 days of culture in αMEM medium. However, this time can vary depending on the individual nuclear donor cells, the particular oocyte and the culture conditions. One skilled in the art will be able to vary the culture conditions desired for optimal growth of a particular embryonic or stem cell-like cell.

The resulting embryonic or stem cell-like cells and cell colonies typically have a more similar appearance to embryonic or stem cell-like cells of the species used as the nucleus cell donor than the donor oocyte species . For example, in the case of embryonic or stem cell-like cells obtained by transferring human nuclear donor cells into enucleated bovine oocytes, the cells exhibit a more similar morphology to mouse embryonic stem cells than bovine ES-like cells. More specifically, the individual cells of a human ES-lineage cell colony are less well defined, and the periphery of the colony is refractive and smooth in appearance. In addition, this cell colony has a long cell doubling time, about twice that of mouse ES cells. Also,
Unlike bovine and porcine-derived ES cells, colonies do not have an epithelial-like appearance.

As discussed above, in US Pat. No. 5,843,780 by Thomson et al., Primate stem cells were SSEA-1 (−), SSEA-4 (+), TRA-1-60 (+), TRA-1 -81 (+) and alkaline phosphatase (+). Human and primate ES cells generated according to the present invention are expected to show similar or identical marker expression. Alternatively, the fact that such cells are in fact human or primate embryonic stem cells will be confirmed based on their ability to give rise to all endodermal, mesodermal and ectodermal tissues. This will be demonstrated by culturing ES cells generated according to the present invention under suitable conditions, for example, the conditions disclosed in Thomson, US Patent No. 5,843,780. The disclosure of this document is included in the present invention. The resulting embryonic or stem cell-like cells and cell lines, preferably human embryonic or stem cell-like cells and cell lines, have numerous therapeutic and diagnostic applications. In particular,
Such embryonic or stem cell-like cells may be used for cell transplant therapy. Human embryonic or stem cell-like cells have application in the treatment of a number of disease states.

From this perspective, it is known that mouse embryonic stem cells (ES cells) can differentiate into almost any cell type, for example hematopoietic stem cells. Therefore, human embryonic or stem cell-like cells produced according to the present invention should have similar differentiation potential. Embryonic or stem cell-like cells according to the invention will be induced to differentiate by known methods to obtain the desired cell type. For example, the subject human embryonic or stem cell-like cells
By culturing such cells in a differentiation medium and under conditions that provide cell differentiation, differentiation may be induced into hematopoietic stem cells, muscle cells, cardiomyocytes, stem cells, chondrocytes, epithelial cells, urinary cells, and the like. Media and methods for effecting embryonic stem cell differentiation are those known in the art as suitable culture conditions. For example, Palacios et al., Proc. Natl. Acad. Sci., USA, 92: 7530-7537 (1995), first cultured stem cell aggregates in a suspension medium lacking retinoic acid and then retinoic acid. Discloses making hematopoietic stem cells from embryonic cell lines by culturing them in the same medium containing and then subjecting the stem cells to an induction procedure that involves transferring cell clumps to a substrate that provides cell adhesion.

Further, Pedersen, J. Reprod. Fertil. Dev., 6: 543-552 (1994) includes various differentiated cell types of embryonic stem cells, especially hematopoietic stem cells, myocytes, cardiomyocytes, neurons. It is a review that references a number of articles that disclose methods for in vitro differentiation into cell types. In addition, Bain et al., Dev. Biol., 168: 342-357 (1995) teaches the in vitro differentiation of embryonic stem cells to produce neurons with neural properties. These documents are examples of the reported methods for obtaining differentiated cells from embryonic or stem cell-like cells. The disclosures of these documents, particularly of the methods for differentiating embryonic stem cells therein, are hereby incorporated by reference in their entirety. Thus, using known methods and media, one of skill in the art can culture the subject embryonic or stem cell-like cells to obtain the desired differentiated cell type, eg, neural cells, muscle cells, hematopoietic stem cells, and the like. Will. In addition, the use of inducible Bcl-2 or Bcl-xl can reduce the viability of certain cell lines.
May be useful to enhance tro outbreaks. In vivo, Bcl-2 inhibits many, but not all, lymphoid cells and many apoptotic cell deaths that occur during neurogenesis. A detailed discussion of how Bcl-2 expression can be used to inhibit apoptosis of related cell lines following transfection of donor cells is disclosed in US Pat. No. 5,646,008. This document is included in the present specification.

The subject embryonic or stem cell-like cells may be used to obtain any desired differentiated cell type. The therapeutic use of such differentiated human cells is unparalleled. For example, human hematopoietic stem cells may be used in medical treatments requiring bone marrow transplants. Such procedures are used to treat many diseases, for example, late stage cancers such as ovarian cancer and leukemia, and diseases that endanger the immune system, such as AIDS. Hematopoietic stem cells can be obtained, for example, by fusing adult somatic cells, such as epithelial cells or lymphocytes, and enucleated oocytes, e.g., bovine oocytes, of a primary or AIDS patient to obtain the embryonic or stem cell-like cells described above. Such cells can be obtained by culturing such cells under conditions suitable for differentiation until hematopoietic stem cells are obtained. Such hematopoietic stem cells may be used to treat cancer and diseases including AIDS.

Alternatively, adult somatic cells of a patient with a neuropathy are fused with enucleated oocytes, eg, primate or bovine oocytes, from which human embryonic or stem cell-like cells are obtained,
Such cells may be cultured under differentiation conditions to produce a neural cell line. Specific diseases that can be treated by transplantation of such human nerve cells include, for example, Parkinson's disease, Alzheimer's disease, ALS and cerebral palsy, among others. Particularly in the case of Parkinson's disease, it has been demonstrated that the transplanted fetal brain nerve cells make proper communication with surrounding cells and produce dopamine. This can cause a long-term reversal of Parkinson's symptoms. To allow for specific selection of differentiated cells, donor cells may be transfected with a selectable marker expressed by an inducible promoter,
This allows selection or enrichment of specific cell lines when differentiation is induced. For example, CD34-neo is used for hematopoietic stem cell selection, Pw1-neo is used for myocyte selection, and Mash-
1-neo may be used to select sympathetic nerve cells, and Mal-neo may be used to select human CNS neurons in gray matter of the cerebral cortex.

A great advantage of the subject invention is that it provides virtually unlimited supply of isogenic or syngenic human cells suitable for transplantation. Thus, the present invention will eliminate an important problem associated with current transplantation methods, namely, rejection of transplanted tissue that may be caused by host versus graft or graft versus host rejection. Traditionally, rejection is prevented or reduced by the administration of anti-rejection agents such as cyclosporine. However, such drugs have significant adverse side effects, such as immunosuppression, carcinogenic properties, and are very expensive. The present invention should eliminate, or at least greatly reduce, the use of anti-rejection agents. Diseases and conditions that can be treated by isogenic cell therapy include, for example, spinal cord injury, multiple sclerosis, muscular dystrophy, diabetes, liver disease, ie, hypercholesterolemia, heart disease, cartilage replacement, burns, leg ulcers, gastrointestinal tract Includes diseases, vascular disease, kidney disease, urinary tract disease and aging associated with disease and health.

Also, human embryonic or stem cell-like cells produced according to the present invention may be used to produce genetically modified or transgenic human differentiated cells. This essentially introduces the desired gene, which may be heterologous,
Alternatively, it may be achieved by removing all or part of the endogenous genes of human embryonic or stem cell-like cells produced according to the present invention so that such cells can differentiate into the desired cell type. . A preferred way to achieve such modifications is by homologous recombination. This is because such techniques can be used to insert, delete or modify genes at specific sites in the stem cell-like cell genome. This methodology is based on defective genes, such as defective immune system genes, cystic fibrosis genes,
Or for the introduction of genes that result in the expression of therapeutically beneficial proteins, such as growth factors, lymphokines, cytokines, enzymes, and the like. For example, genes encoding brain-derived growth factors can be introduced into human embryonic or stem cell-like cells, and cells that have differentiated into neurons can be transplanted into Parkinson's disease patients and lost neurons during such diseases. Delay.

Conventionally, cell types transfected with BDNF range from primary cells to immortal cell lines, and span cells of either neural or non-neural (myoblast and fibroblast) origin. For example, astrocytes can be transformed into BDNF using retroviral vectors.
Transfected with the gene, 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 reduced the Parkinson-like symptoms by 45% in rats 32 days after transplantation (Lundberg et al., Develop. Neurol., 139: 38-53 (1996) and references cited therein). However, such ex vivo systems are problematic. In particular, currently used retroviral vectors are down-regulated in vivo and the transgene is only transiently expressed (reviewed by Mulligan, Science, 226-932 (
1993)). Also, such studies used primary cells, astrocytes, which have a finite survival time and slow self-renewal. Such properties have a deleterious effect on the rate of transfection and prevent the selection of stable transformed cells. Moreover, it is almost impossible to grow large populations of gene-targeted primary cells used in homologous recombination techniques.

In contrast, the difficulties associated with retroviral systems should be eliminated by the use of human embryonic or stem cell-like cells. Previously, the assignee has demonstrated that bovine and porcine embryonic cell lines can be transfected and selected for stable integration of heterologous DNA. Such a method is described in commonly assigned U.S. Ser. No. 08 / 626,054, filed Apr. 1, 1996. This document is included in the present specification. Thus, using such methods, or other known methods, the desired gene may be introduced into the subject embryonic or stem cell-like cells, where the cells are of a desired cell type, such as hematopoietic stem cells, neural cells, etc. Differentiates into cells, pancreatic cells, chondrocytes, etc. Genes that may be introduced into the subject embryonic or stem cell-like cells include, for example, epidermal growth factor, basic fibroblast growth factor, glial-derived neurotrophic growth factor, insulin-like growth factor (I and II), neurotrophy Fin-3, neurotrophin-4 / 5, Syrian neurotrophic factor, AFT-1, cytokine genes (interleukin, interferon, colony stimulating factor, tumor necrosis factor (α, β), etc.), therapeutic enzymes, collagen , A gene encoding human serum albumin, and the like.

In addition, if necessary, one of the negative selection systems known in the art can be used to remove the therapeutic cells from the patient. For example, a donor cell transfected with a thymidine kinase (TK) gene leads to the production of an embryonic cell having the TK gene. The differentiation of these cells also leads to the isolation of therapeutic cells of interest that also express the TK gene. Such cells can be removed from the patient at any time by ganciclovir administration. Such a negative selection system is described in U.S. Patent No. 5,698,446. This document is included in the present specification. The subject embryonic or stem cell-like cells, preferably human cells, may be used as in vitro models of differentiation, and may be used, in particular, for the study of genes involved in early development. Also, differentiated cells, tissues and organs using the subject embryonic or stem cell-like cells may be used for drug research. In addition, the subject embryonic or stem cell-like cells may be used as a nuclear donor for producing other embryonic or stem cell-like cells. The following examples are provided to more clearly describe the subject invention.

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

Nuclear Transfer Procedure The basic nuclear transfer procedure has been previously described. Briefly, slaughterhouse oocytes
After maturation in vitro, approximately 18 hours after maturation (hpm), the oocytes were detached from the cumulus cells and enucleated with a sloped micropipette. For enucleation, use TL-HEPES medium +
Confirmation in bisbenzimide (Hoechst 33342, 3 μg / ml; Sigma). The individual donor cells were then placed in the perivitelline space of the recipient oocyte. Bovine oocyte cytoplasm and donor nuclei (NT units) were fused using electrofusion. One fusion pulse of 90 V, 15 μs was applied to this NT unit. This was done 24 hours after the start of oocyte maturation (hpm). NT units were placed in CR1aa medium to 28 hpm.

A method for artificially activating oocytes has been described elsewhere. NT unit activation was performed at 28hpm. A brief description of this activation procedure is as follows: NT units were ionomycin (5 μM; CalBioch) in TL-HEPES supplemented with 1 mg / ml BSA.
em, La Jolla, CA) for 4 minutes and then TL-HEPES supplemented with 30 mg / ml BSA for 5 minutes
Washed in. Next, this NT unit was transferred to microdroplets of CR1aa medium containing 0.2 mM DMAP (Sigma) and cultured at 38.5 ° C., 5% CO ₂ for 4 to 5 hours. This NT unit was washed and placed in a CR1aa medium supplemented with 10% FCS and 6 mg / ml BSA in a 4-well plate containing a confluent feeder layer of mouse embryo fibroblasts (described below). This NT unit was further cultured at 38.5 ° C. and 5% CO _{2 for} 3 days. The medium was changed every three days until day 12 after activation. At this point, the NT unit that reached the desired cell number, that is, about 50 cells, was mechanically removed from the zone and used for the production of an embryonic cell line. A photograph of the NT unit obtained as described above is included in FIG.

Fibroblast feeder layer Primary cultures of embryonic fibroblasts were obtained from 14-16 day old mouse embryos. After aseptically removing the head, liver, heart and digestive tract, chopped embryos and pre-warmed trypsin
Incubated for 30 minutes at 37 ° C. in EDTA solution (0.05% trypsin / 0.02% EDTA; GIBCO, Grand Island, NY). Put the fibroblasts into a tissue culture flask,
Α-MEM medium (BioWhittaker, Walkersvil, Walkersvil) supplemented with 10% fetal calf serum (FCS) (Hyclone, Logen, UT), penicillin (100 IU / ml) and streptomycin (50 μg / ml).
le, MD). After 3-4 days of passage, (Baxter Sci.
entific, McGaw Park, IL) embryonic fibroblasts were irradiated. Irradiated fibroblasts were grown and maintained at 37 ° C. in a humidified atmosphere containing 5% CO ₂ . This culture dish with uniform monolayer cells was then used to culture embryonic cell lines.

Preparation of Embryonic Cell Line The NT unit cells obtained as described above were washed and directly plated on irradiated feeder fibroblasts. These cells contained cells of the inner part of the NT unit. Cells were harvested with 10% FCS and 0.1 mM β-mercaptoethanol (Sigm
a) was maintained in a growth medium consisting of αMEM. The growth medium was changed every 2-3 days. The first colony was observed on the second day of culture. The colonies grew and showed a morphology similar to the previously disclosed mouse embryonic stem cells (ES cells). The individual cells inside the colony are not very clear, but the periphery of the colony is refractive and smooth in appearance. Colonies appear to have a slower cell doubling time than mouse ES cells. Also, unlike bovine and porcine derived ES cells, this colony has not previously had an epithelial cellular appearance. 2 to 5 are photographs of ES-like cell colonies obtained as described above.

Preparation of Differentiated Human Cells The obtained human embryonic cells were transferred to a differentiation medium and cultured until a differentiated human cell type was observed.

Result

[Table 1] Table 1. Human cells as donor nuclei in NT unit production and development

One NT unit developed into a structure having more than 16 cells was placed on the fibroblast feeder layer. This structure began to grow while forming a colony having an ES cell-like morphology adhered to the feeder layer (for example, see FIG. 2). Furthermore, the 4-16 cell stage structure was not used for the generation of ES cell colonies and for its testing, but this stage has previously been shown to generate ES cells or ES-like cell lines and (Mouse, Eistetter et al., Devel. Grouwth and Differ., 31:27
5-282 (1989); cattle, Stice et al., 1996). Therefore, 4-16 cell stage NT units are also expected to give rise to embryonic or stem cell-like cells and cell colonies. Similar results were obtained by fusion of an adult human keratinocyte cell line and enucleated bovine oocytes cultured in a medium containing ACM, uridine and 1000 IU of LIF. 50
Of the reconstituted embryos, 22 split and one developed to blastocysts on about day 12. The blastocysts were plated and production of ES cell lines was continued.

While the present invention has been described and described with reference to various specific materials, procedures, and examples, the present invention is directed to specific materials, combinations of materials, and procedures selected for that purpose. It is needless to say that the present invention is not limited to this. Various variations of such details will be understood and appreciated by those skilled in the art.

[Brief description of the drawings]

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

FIG. 2 is a photograph of cells for embryonic stem cells derived from the NT unit as shown in FIG.

FIG. 3 is a photograph of cells for embryonic stem cells derived from the NT unit as shown in FIG.

FIG. 4 is a photograph of an NT unit-derived cell for embryonic stem cells as shown in FIG.

FIG. 5 is a photograph of cells for embryonic stem cells derived from the NT unit as shown in FIG.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country 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, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , CA, CH, CN, CU, CZ, DE, DK, 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, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Ruble James Massachusetts 01007 Belchertown Old Enfield 196 ( 72) Inventor Seaberry George, USA 01002 Amherst Village Park, Massachusetts 166 (72) Inventor Steis Steven El, United States of America Massachusetts 01007 Belchertown, Amherst Road 468 F-term (reference) 4B024 AA01 BA21 CA01 DA02 DA03 FA02 GA18 HA01 4B090 A90 AAX AB01 AB04 AB05 AB06 AB08 AC20 BA01 BA08 CA27 CA44 CA60

Claims (50)

[Claims] 1. A method for producing embryonic or stem cell-like cells, comprising: (i) inserting a desired differentiated human cell or mammalian cell or cell nucleus into an enucleated animal oocyte. The oocyte is derived from a species different from human or mammalian cells under conditions suitable for the formation of a nuclear transfer (NT) unit; (ii) activating the resulting nuclear transfer unit (Iii) culturing the activated nuclear transfer unit until it becomes larger than the two-cell development stage; and (iv) culturing cells obtained from the cultured NT unit to obtain embryonic or stem cell-like cells. Obtaining the cell line. 2. A cell inserted into an enucleated animal oocyte is a human cell.
The method of claim 1. 3. The method according to claim 2, wherein the human cells are adult cells. 4. The method according to claim 2, wherein the human cells are epithelial cells, keratinocytes, lymphocytes or fibroblasts. 5. The method according to claim 2, wherein the oocyte is obtained from a mammal. 6. The method of claim 5, wherein the animal oocyte is obtained from an ungulate. 7. The method according to claim 6, wherein the ungulate is selected from the group consisting of cows, sheep, pigs, horses, goats, and buffaloes. 8. The method of claim 1, wherein the enucleated cells have been matured prior to enucleation.
The method described in. 9. The method of claim 1, wherein the fusion nuclear transfer unit is activated in vitro. 10. The method according to claim 1, wherein the activated nuclear transfer unit is cultured on a culture feeder layer. 11. The method according to claim 10, wherein the feeder layer comprises fibroblasts. 12. The method according to claim 1, wherein the NT unit having 16 or more cells in (iv) is cultured on the feeder cell layer. 13. The method of claim 12, wherein the feeder cell layer comprises fibroblasts. 14. The method of claim 13, wherein the fibroblasts comprise mouse embryonic fibroblasts. 15. The method according to claim 1, wherein the obtained embryonic or stem cell-like cells are induced to differentiate. 16. The method according to claim 2, wherein the obtained embryonic or ring cell-like cells are induced to differentiate. 17. The method of claim 1, wherein the fusion is performed by electrofusion. 18. An embryonic or stem cell-like cell obtained by the method according to claim 1. 19. A human embryonic or stem cell-like cell obtained by the method according to claim 2. 20. A human embryonic or stem cell-like cell obtained by the method according to claim 3. 21. A human embryonic or stem cell-like cell obtained by the method of claim 4. 22. A human embryonic or stem cell-like cell obtained by the method according to claim 6. 23. A human embryonic or stem cell-like cell obtained by the method of claim 7. 24. A differentiated human cell obtained by the method according to claim 16. 25. The method according to claim 24, which is selected from the group consisting of nerve cells, hematopoietic stem cells, pancreatic cells, muscle cells, chondrocytes, urinary cells, hepatocytes, germ cells, skin cells, intestinal cells, and gastric cells. Differentiated human cells. 26. A method for treating, comprising administering to a patient in need of cell transplantation therapy the isogenically differentiated human cell according to claim 24. 27. The cell transplantation treatment is performed for Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, spinal cord disease or injury, multiple sclerosis, muscular dystrophy, cystic fibrosis, liver disease, diabetes, heart disease, cartilage disease or injury. 27. The method of claim 26, wherein the method is performed to treat a disease or condition selected from the group consisting of: burn, leg ulcer, vascular disease, urinary tract disease, AIDS and cancer. 28. The method according to claim 26, wherein the differentiated human cells are hematopoietic stem cells or neural cells. 29. The method of claim 26, wherein the treatment is for Parkinson's disease and the differentiated cells are neurons. 30. The method of claim 26, wherein the treatment is for cancer and the differentiated cells are hematopoietic stem cells. 31. The differentiated human cell of claim 24, comprising and expressing an inserted gene. 32. The method of claim 1, wherein the desired gene is inserted, removed or modified in embryonic or stem cell-like cells. 33. The method of claim 32, wherein the desired gene is a gene encoding a therapeutic enzyme, growth factor or cytokine. 34. The method of claim 32, wherein the embryonic or stem cell-like cells are human embryonic or stem cell-like cells. 35. The method according to claim 32, wherein the desired gene is removed, modified or deleted by homologous recombination. 36. The method of claim 1, wherein the donor cells are genetically modified to inhibit the development of at least one of endoderm, mesoderm, and ectoderm. 37. The donor cell is modified to increase differentiation efficiency,
The method of claim 1. 38. The method according to claim 36, wherein the cultured nuclear transfer unit is cultured in a medium containing at least one caspase inhibitor. 39. The method of claim 1, wherein the donor cells express a detectable label indicative of the expression of a particular cyclin. 40. The donor cell is a gene selected from the group consisting of SRF, MESP-1, HNF-4, beta-1, integrin, MSD, GATA-6, GATA-4, RNA helicase A and Hbeta58. 37. The method of claim 36, wherein the method has been modified to alter expression of 41. The method of claim 37, wherein the donor cell has been genetically modified by introducing a DNA that confers expression of Q7 and / or Q9. 42. The method of claim 41, wherein the gene is operably linked to a controllable promoter. 43. The method of claim 1, wherein the donor cells have been genetically modified to inhibit apoptosis. 44. The reduction in apoptosis is indicated by Bad, Bok, BH3, Bik, Blk, Hrk,
BNIP3, Gim _L , Bid, BGL-1, Bcl-XL, Bcl-w, Mcl-1, A1, Nr-13, BHRF-1, LMW5-H
44. The method of claim 43, wherein the method is provided by altering the expression of one or more genes selected from the group consisting of L, ORF16, Ks-Bcl-2, E1B-19K, and CED-9. 45. The method of claim 44, wherein the gene is operably connected to an inducible promoter. 46. A mammalian somatic cell that expresses a DNA encoding a detectable marker, wherein the expression of the DNA is linked to a specific cyclin. 47. The cell of claim 46, wherein the cyclin is selected from the group consisting of cyclins D1, D2, D3, B1, B2, E, A and H. 48. The detectable marker is a fluorescent polypeptide.
7. The cell according to 6. 49. The mammal cell may be a human cell, a primate cell, a rodent cell,
49. The cell according to claim 48, wherein the cell is selected from the group consisting of ungulate cells, dog cells, and cat cells. 50. The cell of claim 4, which is a human cell, a bovine cell, or a primate cell.
9. The cell according to 8.