AU759322B2

AU759322B2 - Embryonic or stem-like cell lines produced by cross-species nuclear transplantation

Info

Publication number: AU759322B2
Application number: AU29795/99A
Authority: AU
Inventors: Jose Cibelli; James Robl; Steven L. Stice
Original assignee: University of Massachusetts Amherst
Current assignee: University of Massachusetts Amherst
Priority date: 1998-03-02
Filing date: 1999-03-02
Publication date: 2003-04-10
Anticipated expiration: 2019-03-02
Also published as: EP1060243A4; WO1999045100A1; EP1060243A1; JP2002505100A; BR9908499A; CN1299408A; AU2979599A; IL138201A0; NZ506808A; CA2323094A1

Description

WO 99/45100 PCT/US99/04608 -1- 1 EMBRYONIC OR STEM-LIKE CELL LINES PRODUCED BY 2 CROSS SPECIES NUCLEAR TRANSPLANTATION 3 4 6 7 FIELD OF THE INVENTION 8 The present invention generally relates to the production of embryonic or 9 stem-like cells by transplantation of cell nuclei derived from animal or human cells into enucleated animal oocytes of a species different from the donor nuclei.

11 The present invention more specifically relates to the production of primate or 12 human embryonic or stem-like cells by transplantation of the nucleus of a 13 primate or human cell into an enucleated animal oocyte, a primate or 14 ungulate oocyte and in a preferred embodiment a bovine enucleated oocyte.

The present invention further relates to the use of the resultant embryonic 16 or stem-like cells, preferably primate or human embryonic or stem-like cells for 17 therapy, for diagnostic applications, for the production of differentiated cells 18 which may also be used for therapy or diagnosis, and for the production of 19 transgenic embryonic or transgenic differentiated cells, cell lines, tissues and organs. Also, the embryonic or stem-like cells obtained according to the present 21 invention may themselves be used as nuclear donors in nuclear transplantation 22 or nuclear transfer methods for the production of chimeras or clons, preferably 23 transgenic cloned or chimeric animals.

24 BACKGROUND OF THE INVENTION Methods for deriving embryonic stem (ES) cell lines in vitro from early 26 preimplantation mouse embryos are well known. (See, Evans et al., Nature, .WO 99/45100 PCT/US99/04608 -2- 1 29:154-156 (1981); Martin, Proc. Natl. Acad. Sci., USA, 78:7634-7638 (1981)).

2 ES cells can be passaged in an undifferentiated state, provided that a feeder layer 3 of fibroblast cells (Evans et al., Id.) or a differentiation inhibiting source (Smith 4 et al., Dev. Biol., 121:1-9 (1987)) is present.

ES cells have been previously reported to possess numerous applications.

6 For example, it has been reported that ES cells can be used as an in vitro model 7 for differentiation, especially for the study of genes which are involved in the 8 regulation of early development. Mouse ES cells can give rise to germline 9 chimeras when introduced into preimplantation mouse embryos, thus demonstrating their pluripotency (Bradley et al., Nature, 309:255-256 (1984)).

11 In view of their ability to transfer their genome to the next generation, ES 12 cells have potential utility for germline manipulation of livestock animals by 13 using ES cells with or without a desired genetic modification. Moreover, in the 14 case of livestock animals, ungulates, nuclei from like preimplantation livestock embryos support the development of enucleated oocytes to term (Smith 16 et al., Biol. Reprod., 40:1027-1035 (1989); and Keefer et al., Biol. Reprod., 17 50:935-939 (1994)). This is in contrast to nuclei from mouse embryos which 18 beyond the eight-cell stage after transfer reportedly do not support the 19 development of enucleated oocytes (Cheong et al, Biol. Reprod., 48:958 (1993)).

Therefore, ES cells from livestock animals are highly desirable because they may 21 provide a potential source of totipotent donor nuclei, genetically manipulated or 22 otherwise, for nuclear transfer procedures.

23 Some research groups have reported the isolation of purportedly 24 pluripotent embryonic cell lines. For example, Notarianni et al., J. Reprod. Fert.

Suppl., 43:255-260 (1991), report the establishment of purportedly stable, 26 pluripotent cell lines from pig and sheep blastocysts which exhibit some WO 99/45100 PCT/US99/04608 -3- 1 morphological and growth characteristics similar to that of cells in primary 2 cultures of inner cell masses isolated immunosurgically from sheep blastocysts.

3 Also, Notarianni et al., J Reprod. Fert. Suppl., 41:51-56 (1990) discloses 4 maintenance and differentiation in culture of putative pluripotential embryonic cell lines from pig blastocysts. Further, Gerfen et al., Anim. Biotech, 6(1):1-14 6 (1995) disclose the isolation of embryonic cell lines from porcine blastocysts.

7 These cells are stably maintained in mouse embryonic fibroblast feeder layers 8 without the use of conditioned medium. These cells reportedly differentiate into 9 several different cell types during culture (Gerfen et al., Id.).

Further, Saito et al., Roux's Arch. Dev. Biol., 201:134-141 (1992) report 11 bovine embryonic stem cell-like cell lines cultured which survived passages for 12 three, but were lost after the fourth passage. Still further, Handyside et al., 13 Roux's Arch. Dev. Biol., 196:185-190 (1987) disclose culturing ofimmunosurgi- 14 cally isolated inner cell masses of sheep embryos under conditions which allow for the isolation of mouse ES cell lines derived from mouse ICMs. Handyside 16 et al. (1987) report that under such conditions, the sheep ICMs attach, 17 spread, and develop areas of both ES cell-like and endoderm-like cells, but that 18 after prolonged culture only endoderm-like cells are evident. (Id.) 19 Recently, Cherny et al., Theriogenology, 41:175 (1994) reported purportedly pluripotent bovine primordial germ cell-derived cell lines maintained 21 in long-term culture. These cells, after approximately seven days in culture, 22 produced ES-like colonies which stain positive for alkaline phosphatase (AP), 23 exhibited the ability to form embryoid bodies, and spontaneously differentiated 24 into at least two different cell types. These cells also reportedly expressed mRNA for the transcription factors OCT4, OCT6 and HES1, a pattern of 26 homeobox genes which is believed to be expressed by ES cells exclusively.

.WO 99/45100 PCT/US99/04608 -4- 1 Also recently, Campbell et al., Nature, 380:64-68 (1996) reported the 2 production of live lambs following nuclear transfer of cultured embryonic disc 3 (ED) cells from day nine ovine embryos cultured under conditions which 4 promote the isolation of ES cell lines in the mouse. The authors concluded based on their results that ED cells from day nine ovine embryos are totipotent by 6 nuclear transfer and that totipotency is maintained in culture.

7 Van Stekelenburg-Hamers et al., Mol. Reprod. Dev., 40:444-454 (1995), 8 reported the isolation and characterization of purportedly permanent cell lines 9 from inner cell mass cells of bovine blastocysts. The authors isolated and cultured ICMs from 8 or 9 day bovine blastocysts under different conditions to 11 determine which feeder cells and culture media are most efficient in supporting 12 the attachment and outgrowth of bovine ICM cells. They concluded based on 13 their results that the attachment and outgrowth of cultured ICM cells is enhanced 14 by the use of STO (mouse fibroblast) feeder cells (instead of bovine uterus epithelial cells) and by the use of charcoal-stripped serum (rather than normal se- 16 rum) to supplement the culture medium. Van Stekelenburg et al reported, 17 however, that their cell lines resembled epithelial cells more than pluripotent 18 ICM cells. (Id.) 19 Still further, Smith et al., WO 94/24274, published October 27, 1994, Evans et al, WO 90/03432, published April 5, 1990, and Wheeler et al, WO 21 94/26889, published November 24, 1994, report the isolation, selection and 22 propagation of animal stem cells which purportedly may be used to obtain 23 transgenic animals. Also, Evans et al., WO 90/03432, published on April 24 1990, reported the derivation of purportedly pluripotent embryonic stem cells derived from porcine and bovine species which assertedly are useful for the 26 production of transgenic animals. Further, Wheeler et al, WO 94/26884, WO 99/45100 PCT/US99/04608 1 published November 24, 1994, disclosed embryonic stem cells which are 2 assertedly useful for the manufacture of chimeric and transgenic ungulates.

3 Thus, based on the foregoing, it is evident that many groups have attempted to 4 produce ES cell lines, because of their potential application in the production of cloned or transgenic embryos and in nuclear transplantation.

6 The use of ungulate ICM cells for nuclear transplantation has also been 7 reported. For example, Collas et al., Mol. Reprod. Dev., 38:264-267 (1994) 8 disclose nuclear transplantation of bovine ICMs by microinjection of the lysed 9 donor cells into enucleated mature oocytes. The reference disclosed culturing of embryos in vitro for seven days to produce fifteen blastocysts which, upon 11 transferral into bovine recipients, resulted in four pregnancies and two births.

12 Also, Keefer et al., Biol. Reprod., 50:935-939 (1994), disclose the use of bovine 13 ICM cells as donor nuclei in nuclear transfer procedures, to produce blastocysts 14 which, upon transplantation into bovine recipients, resulted in several live offspring. Further, Sims et al., Proc. Natl. Acad. Sci., USA, 90:6143-6147 16 (1993), disclosed the production of calves by transfer of nuclei from short-term 17 in vitro cultured bovine ICM cells into enucleated mature oocytes.

18 Also, the production of live lambs following nuclear transfer of cultured 19 embryonic disc cells has been reported (Campbell et al., Nature, 380:64-68 (1996)). Still further, the use of bovine pluripotent embryonic cells in nuclear 21 transfer and the production of chimeric fetuses has also been reported (Stice 22 et al., Biol. Reprod., 54:100-110 (1996)); Collas et al, Mol. Reprod. Dev., 23 38:264-267 (1994).

24 Also, there have been previous attempts to produce cross species NT units (Wolfe et al., Theriogenology, 33:350 (1990). Specifically, bovine embryonic 26 cells were fused with bison oocytes to produce some cross species NT units .WO 99/45100 PCT/US99/04608 -6- 1 possibly having an inner cell mass. However, embryonic cells, not adult cells 2 were used, as donor nuclei in the nuclear transfer procedure. The dogma has 3 been that embryonic cells are more easily reprogrammed than adult cells. This 4 dates back to earlier NT studies in the frog (review by DiBerardino, Differentiation, 17:17-30 (1980)). Also, this study involved very 6 phylogenetically similar animals (cattle nuclei and bison oocytes). By contrast, 7 previously when more diverse species were fused during NT (cattle nuclei into 8 hamster oocytes), no inner cell mass structures were obtained. Further, it has 9 never been previously reported that the inner cell mass cells from NT units could be used to form an ES cell-like colony that could be propagated.

11 Also, Collas et al taught the use of granulosa cells (adult somatic 12 cells) to produce bovine nuclear transfer embryos. However, unlike the present 13 invention, these experiments did not involve cross-species nuclear transfer.

14 Also, unlike the present invention ES-like cell colonies were not obtained.

Very recently, U.S. Patent No. 5,843,780, issued to James A. Thomson 16 on December 1, 1998, assigned to the Wisconsin Alumni Research Foundation, 17 that purports to disclose a purified preparation of primate embryonic stem cells 18 that are capable of proliferation in an in vitro culture for over one year; (ii) 19 maintain a karyotype in which all chromosomes characteristic of the primate species are present and not noticeably altered through prolonged culture; (iii) 21 maintains the potential to differentiate into derivatives of endoderm, mesoderm 22 and ectoderm tissues throughout culture; and (iv) will not differentiate when 23 cultured on a fibroblast feeder layer. These cells were reportedly negative for the 24 SSEA-1 marker, positive for the SEA-3 marker, positive for the SSEA-4 marker, express alkaline phosphatase activity, are pluripotent, and have karyotypes which 26 include the presence of all the chromosomes characteristic of the primate species WO 99/45100 PCT/US99/04608 -7- 1 and in which none of the chromosomes are altered. Further, these cells are 2 respectfully positive for the TRA-1-60, and TRA-1-81 markers. The cells 3 purportedly differentiate into endoderm, mesoderm and ectoderm cells when 4 injected into a SCID mouse. Also, purported embryonic stem cell lines derived from human or primate blastocytes are discussed in Thomson et al., Science 6 282:1145-1147 and Proc. Natl. Acad. Sci., USA 92:7844-7848 (1995).

7 Thus, Thomson disclose what purportedly are non-human primate and 8 human embryonic or stem-like cells and methods for their production. However, 9 there still exists a significant need for methods for producing human embryonic or stem-like cells that are autologous to an intended transplant recipient given 11 their significant therapeutic and diagnostic potential.

12 In this regard, numerous human diseases have been identified which may 13 be treated by cell transplantation. For example, Parkinson's disease is caused by 14 degeneration of dopaminergic neurons in the substantia nigra. Standard treatment for Parkinson's involves administration of L-DOPA, which temporarily 16 ameliorates the loss of dopamine, but causes severe side effects and ultimately 17 does not reverse the progress of the disease. A different approach to treating 18 Parkinson's, which promises to have broad applicability to treatment of many 19 brain diseases and central nervous system injury, involves transplantation of cells or tissues from fetal or neonatal animals into the adult brain. Fetal neurons from 21 a variety of brain regions can be incorporated into the adult brain. Such grafts 22 have been shown to alleviate experimentally induced behavioral deficits, includ- 23 ing complex cognitive functions, in laboratory animals. Initial test results from 24 human clinical trials have also been promising. However, supplies of human fetal cells or tissue obtained from miscarriages is very limited. Moreover, 26 obtaining cells or tissues from aborted fetuses is highly controversial.

11 -8- 1 There is currently no available procedure for producing "fetal-like" cells 2 from the patient. Further, allograft tissue is not readily available and both allo- 3 graft and xenograft tissue are subject to graft rejection. Moreover, in some cases, 4 it would be beneficial to make genetic modifications in cells or tissues before transplantation. However, many cells or tissues wherein such modification 6 would be desirable do not divide well in culture and most types of genetic 7 transformation require rapidly dividing cells.

8 There is therefore a clear need in the art for a supply of human embryonic 9 or stem-like undifferentiated cells for use in transplants and cell and gene therapies.

11 OBJECTS OF THE INVENTION 12 It is an object of the invention to provide novel and improved methods for 13 producing embryonic or stem-like cells.

14 It is a more specific object of the invention to provide a novel method for producing embryonic or stem-like cells which involves transplantation of the S 16 nucleus of a mammalian or human cell into an enucleated oocyte of a different 17 species.

1. It is preferable that the invention provides a novel method for :19 producing non-human primate or human embryonic or stem-like cells which 20 involves transplantation of the nucleus of a non-human primate or human cell 21 into an enucleated animal or human oocyte, an ungulate, human or primate 22 enucleated oocyte.

23 It is also preferable that the invention provides a novel method for 24 producing lineage-defective non-human primate or human embryonic or sternlike cells which involves transplantation of the nucleus of a non-human primate 26 or human cell, a human adult cell into an enucleated non-human primate or -9- 1 human oocyte, wherein such cell has been genetically engineered to be incapable 2 of differentiation into a specific cell lineage or has been modified such that the 3 cells are "mortal", and thereby do not give rise to a viable offspring, by 4 engineering expression of anti-sense or ribozyme telomerase gene.

It is preferable that the invention enhances efficiency of nuclear 6 transfer and specifically to enhance the development ofpreimplantation embryos 7 produced by nuclear transfer by genetically engineering donor somatic cells used 8 for nuclear transfer to provide for the expression of genes that enhance 9 embryonic development, genes of the MHC I family, and in particular Ped genes such as Q7 and/or Q9.

11 It is further preferable that the invention enhances the production of S: 12 nuclear transfer embryos by IVP and more specifically nuclear transfer embryos 13 by genetically altering the donor cell used for nuclear transfer such that it is 14 resistant to apoptosis, by introduction of a DNA construct that provides for S15 the expression of genes that inhibit apoptosis, Bcl-2 or Bcl-2 family 16 members and/or by the expression of antisense ribozymes specific to genes that S. 17 induce apoptosis during early embryonic development.

:e 18 It is also preferable that the invention improves the efficacy of nuclear 19 transfer by improved selection of donor cells of a specific cell cycle stage, e.g., 20 G phase, by genetically engineering donor cells such that they express a DNA S" 21 construct encoding a particular cyclin linked to a detectable marker, one that 22 encodes a visualizable fluorescent tag) marker protein.

23 It is also preferable that the invention enhances the development of in vitro 24 produced embryos, by culturing such embryos in the presence of one or more protease inhibitors, preferably one or more caspase inhibitors, thereby inhibiting 26 apoptosis.

It is preferable that the invention provides embryonic or stem-like cells produced by transplantation of nucleus of an animal or human cell into an enucleated oocyte of a different species.

It is further preferable that the invention provides primate or human embryonic or stem-like cells produced by transplantation of the nucleus of a primate or human cell into an enucleated animal oocyte, a human, primate or ungulate enucleated oocyte.

It is also preferable that according to the invention such embryonic or stem-like cells are used for therapy or diagnosis.

It is preferable that according to the invention such primate or human embryonic or stem-like cells are used for treatment or diagnosis of any disease wherein cell, tissue or organ transplantation is therapeutically or diagnostically beneficial.

It is preferable that according to the invention the embryonic or stem-like cells produced according to the invention are used for the production of differentiated cells, .tissues or organs.

15 It is more preferable that according to the invention the primate or human embryonic or stem-like cells produced according to the invention are used for the production of differentiated human cells, tissues or organs.

It is also preferable that according to the invention the embryonic or stem-like cells produced according to the invention are used for the production of genetically S 20 engineered embryonic or stem-like cells, which cells may be used to produce genetically o• .;Z engineered or transgenic differentiated human cells, tissues or organs, having use in gene therapies.

It is preferable that according to the invention the embryonic or stem-like cells produced according to the invention are used in vitro, e.g. for study of cell differentiation 2 and for assay purposes, e.g. for drug studies.

11 It is preferable that the invention provides improved methods of transplantation therapy, comprising the usage of isogenic or synegenic cells, tissues or organs produced from the embryonic or stem-like cells produced according to the invention. Such therapies include by way of example treatment of diseases and injuries including Parkinson's, Huntington's, Alzheimer's, ALS, spinal cord injuries, multiple sclerosis, muscular dystrophy, diabetes, liver diseases, heart disease, cartilage replacement, bums, vascular diseases, urinary tract diseases, as well as for the treatment of immune defects, bone marrow transplantation, cancer, among other diseases.

It is preferable that the transgenic or genetically engineered embryonic or stemlike cells produced according to the invention are used for gene therapy, in particular for the treatment and/or prevention of the diseases and injuries identified, supra.

It is also preferable that the embryonic or stem-like cells produced according to the invention or transgenic or genetically engineered embryonic or stem-like cells produced according to the invention are used as nuclear donors for nuclear S" 15 transplantation.

It is further preferable that the genetically engineered ES cells produced according .to the invention are used for the production of transgenic animals, non-human primates, rodents, ungulates, etc. Such transgenic animals can be used to produce, e.g., animal models for study of human diseases, or for the production of desired polypeptides, S 20 therapeutics or nutripharmaceuticals.

With the foregoing and other objects, advantages and preferred features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the preferred embodiments of the invention and to the appended claims.

12 BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a photograph of a nuclear transfer (NT) unit produced by transfer of an adult human cell into an enucleated bovine oocyte.

Figures 2 to 5 are photographs of embryonic stem-like cells derived from a NT unit such as is depicted in Figure 1.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel method for producing embryonic or stem-like cells, and more specifically non-human primate or human embryonic or stem-like cells by nuclear transfer or nuclear transplantation. In the subject application, nuclear transfer or nuclear transplantation or NT are used interchangeably.

As discussed supra, the isolation of actual embryonic or stem-like cells by nuclear transfer or nuclear transplantation has never been reported. Rather, previous reported isolation of ES-like cells has been from fertilized embryos.

Also, successful nuclear transfer involving cells or DNA of genetically dissimilar species, or more specifically adult cells or DNA of one species human) and oocytes of another non-related species has never been reported. Rather, while 15 embryos produced by fusion of cells of closely related species, has been reported, bovine-goat and bovine-bison, they did not produce ES cells. (Wolfe et al, Theriogenology, 33(1):350 (1990).) Also, there has never been reported a method for producing primate or human ES cells derived from a non-fetal tissue source. Rather, the limited human fetal cells and tissues which are currently available must be obtained or derived from spontaneous abortion tissues and 20 from aborted fetuses.

Also, prior to the present invention, no one obtained embryonic or stem- WO 99/45100 PCT/US99/04608 -13- 1 like cells by cross-species nuclear transplantation.

2 Quite unexpectedly, the present inventors discovered that human 3 embryonic or stem-like cells and cell colonies may be obtained by 4 transplantation of the nucleus of a human cell, an adult differentiated human cell, into an enucleated animal oocyte, which is used to produce nuclear transfer 6 (NT) units, the cells of which upon culturing give rise to human embryonic or 7 stem-like cells and cell colonies. This result is highly surprising because it is the 8 first demonstration of effective cross-species nuclear transplantation involving 9 the introduction of a differentiated donor cell or nucleus into an enucleated oocyte of a genetically dissimilar species, the transplantation of cell nuclei 11 from a differentiated animal or human cell, adult cell, into the enucleated 12 egg of a different animal species, to produce nuclear transfer units containing 13 cells which when cultured under appropriate conditions give rise to embryonic 14 or stem-like cells and cell colonies.

Preferably, the NT units used to produce ES-like cells will be cultured to 16 a size of at least 2 to 400 cells, preferably 4 to 128 cells, and most preferably to 17 a size of at least about 50 cells.

18 In the present invention, embryonic or stem-like cells refer to cells 19 produced according to the present invention. The present application refers to such cells as stem-like cells rather than stem cells because of the manner in 21 which they are typically produced, by cross-species nuclear transfer. While 22 these cells are expected to possess similar differentiation capacity as normal stem 23 cells they may possess some insignificant differences because of the manner they 24 are produced. For example, these stem-like cells may possess the mitochondria of the oocytes used for nuclear transfer, and thus not behave identically to 26 conventional embryonic stem cells.

WO 99/45100 PCT/US99/04608 -14- 1 The present discovery was made based on the observation that nuclear 2 transplantation of the nucleus of an adult human cell, specifically a human 3 epithelial cell obtained from the oral cavity of a human donor, when transferred 4 into an enucleated bovine oocyte, resulted in the formation of nuclear transfer units, the cells of which upon culturing gave rise to human stem-like or embry- 6 onic cells and human embryonic or stem-like cell colonies. This result has 7 recently been reproduced by transplantation of keratinocytes from an adult 8 human into an enucleated bovine oocyte with the successful production of a 9 blastocyst and ES cell line. Based thereon, it is hypothesized by the present inventors that bovine oocytes and human oocytes, and likely mammals in general 11 must undergo maturation processes during embryonic development which are 12 sufficiently similar or conserved so as to permit the bovine oocyte to function as 13 an effective substitute or surrogate for a human oocyte. Apparently, oocytes in 14 general comprise factors, likely proteinaceous or nucleic acid in nature, that induce embryonic development under appropriate conditions, and these functions 16 that are the same or very similar in different species. These factors may 17 comprise material RNAs and/or telomerase.

18 Based on the fact that human cell nuclei can be effectively transplanted 19 into bovine oocytes, it is reasonable to expect that human cells may be 2 transplanted into oocytes of other non-related species, other ungulates as 21 well as other animals. In particular, other ungulate oocytes should be suitable, 22 e.g. pigs, sheep, horses, goats, etc. Also, oocytes from other sources should be 23 suitable, e.g. oocytes derived from other primates, amphibians, rodents, rabbits, 24 guinea pigs, etc. Further, using similar methods, it should be possible to transfer human cells or cell nuclei into human oocytes and use the resultant blastocytes 26 to produce human ES cells.

WO 99/45100 PCT/US99/04608 1 Therefore, in its broadest embodiment, the present invention involves the 2 transplantation of an animal or human cell nucleus or animal or human cell into 3 the enucleated oocyte of an animal species different from the donor nuclei, by 4 injection or fusion, to produce an NT unit containing cells which may be used to obtain embryonic or stem-like cells and/or cell cultures. For example, the 6 invention may involve the transplantation of an ungulate cell nucleus or ungulate 7 cell into an enucleated oocyte of another species, another ungulate or non- 8 ungulate, by injection or fusion, which cells and/or nuclei are combined to 9 produce NT units and which are cultured under conditions suitable to obtain multicellular NT units, preferably comprising at least about 2 to 400 cells, more 11 preferably 4 to 128 cells, and most preferably at least about 50 cells. The cells 12 of such NT units may be used to produce embryonic or stem-like cells or cell 13 colonies upon culturing.

14 However, the preferred embodiment of the invention comprises the production of non-human primate or human embryonic or stem-like cells by 16 transplantation of the nucleus of a donor human cell or a human cell into an 17 enucleated human, primate, or non-primate animal oocyte, an ungulate 18 oocyte, and in a preferred embodiment a bovine enucleated oocyte.

19 In general, the embryonic or stem-like cells will be produced by a nuclear transfer process comprising the following steps: 21 obtaining desired human or animal cells to be used as a source of 22 donor nuclei (which may be genetically altered); 23 (ii) obtaining oocytes from a suitable source, e.g. a mammal and most 24 preferably a primate or an ungulate source, e.g. bovine, (iii) enucleating said oocytes; 26 (iv) transferring the human or animal cell or nucleus into the enucleated WO 99/45100 PCT/US99/04608 -16- 1 oocyte of an animal species different than the donor cell or nuclei, by fusion 2 or injection; 3 culturing the resultant NT product or NT unit to produce multiple cell 4 structures; and (vi) culturing cells obtained from said embryos to obtain embryonic or 6 stem-like cells and stem-like cell colonies.

7 Nuclear transfer techniques or nuclear transplantation techniques are 8 known in the literature and are described in many of the references cited in the 9 Background of the Invention. See, in particular, Campbell et al, Theriogenology, 43:181 (1995); Collas et al, Mol. Report Dev., 38:264-267 (1994); Keefer et al, 11 Biol. Reprod., 50:935-939 (1994); Sims et al, Proc. Natl. Acad. Sci., USA, 12 90:6143-6147 (1993); WO 94/26884; WO 94/24274, and WO 90/03432, which 13 are incorporated by reference in their entirety herein. Also, U.S. Patent Nos.

14 4,944,384 and 5,057,420 describe procedures for bovine nuclear transplantation.

See, also Cibelli et al, Science, Vol. 280:1256-1258 (1998).

16 Human or animal cells, preferably mammalian cells, may be obtained and 17 cultured by well known methods. Human and animal cells useful in the present 18 invention include, by way of example, epithelial, neural cells, epidermal cells, 19 keratinocytes, hematopoietic cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), other immune cells, erythrocytes, macrophages, 21 melanocytes, monocytes, mononuclear cells, fibroblasts, cardiac muscle cells, 22 and other muscle cells, etc. Moreover, the human cells used for nuclear transfer 23 may be obtained from different organs, skin, lung, pancreas, liver, stomach, 24 intestine, heart, reproductive organs, bladder, kidney, urethra and other urinary organs, etc. These are just examples of suitable donor cells. Suitable donor 26 cells, cells useful in the subject invention, may be obtained from any cell or WO 99/45100 PCT/US99/04608 -17- 1 organ of the body. This includes all somatic or germ cells. Preferably, the donor 2 cells or nucleus would comprise actively dividing, non-quiescent, cells as 3 this has been reported to enhance cloning efficacy. Also preferably, such donor 4 cells will be in the G1 cell cycle.

The resultant blastocytes may be used to obtain embryonic stem cell lines 6 according to the culturing methods reported by Thomson et al., Science 7 282:1145-1147 (1998) and Thomson et al., Proc. Natl. Acad. Sci., USA 92:7544- 8 7848 (1995), incorporated by reference in their entirety herein.

9 In the example which follows the cells used as donors for nuclear transfer were epithelial cells derived from the oral cavity of a human donor and adult 11 human keratinocytes. However, as discussed, the disclosed method is applicable 12 to other human cells or nuclei. Moreover, the cell nuclei may be obtained from 13 both human somatic and germ cells.

14 It is also possible to arrest donor cells at mitosis before nuclear transfer, using a suitable technique known in the art. Methods for stopping the cell cycle 16 at various stages have been thoroughly reviewed in U.S. Patent 5,262,409, which 17 is herein incorporated by reference. In particular, while cycloheximide has been 18 reported to have an inhibitory effect on mitosis (Bowen and Wilson (1955) J.

19 Heredity 45: it may also be employed for improved activation of mature bovine follicular oocytes when combined with electric pulse treatment (Yang et 21 al. (1992) Biol. Reprod. 42 (Suppl. 117).

22 Zygote gene activation is associated with hyperacetylation ofHistone H4.

23 Trichostatin-A has been shown to inhibit histone deacetylase in a reversible 24 manner (Adenot et al. Differential H4 acetylation of paternal and maternal chromatin precedes DNA replication and differential transcriptional activity in 26 pronuclei of 1-cell mouse embryos. Development (Nov. 1997) 124(22): 4615- WO 99/45100 PCT/US99/04608 -18- 1 4625; Yoshida et al. Trichostatin A and trapoxin: novel chemical probes for the 2 role ofhistone acetylation in chromatin structure and function. Bioessays (May, 3 1995) 17(5): 423-430), as have other compounds.

4 For instance, butyrate is also believed to cause hyper-acetylations of histones by inhibiting histone deacetylase. Generally, butyrate appears to modify 6 gene expression and in almost all cases its addition to cells in culture appears to 7 arrest cell growth. Use of butyrate in this regard is described in U.S. Patent No.

8 5,681,718, which is herein incorporated by reference. Thus, donor cells may be 9 exposed to Trichostatin-A or another appropriate deacetylase inhibitor prior to fusion, or such a compound may be added to the culture media prior to genome 11 activation.

12 Additionally, demethylation of DNA is thought to be a requirement for 13 proper access of transcription factors to DNA regulatory sequences. Global 14 demethylation of DNA from the eight-cell stage to the blastocyst stage in preimplantation embryos has previously been described (Stein et al., Mol.

16 Reprod. Dev. 47(4): 421-429). Also, Jaenisch et al. (1997) have reported that 17 5-azacytidine can be used to reduce the level of DNA methylation in cells, 18 potentially leading to increased access of transcription factors to DNA regulatory 19 sequences. Accordingly, donor cells may be exposed to 5-azacytidine previous to fusion, or 5-Aza may be added to the culture medium from the 8 cell 21 stage to blastocyst. Alternatively, other known methods for effecting DNA 22 demethylation may be used.

23 The oocytes used for nuclear transfer may be obtained from animals 24 including mammals and amphibians. Suitable mammalian sources for oocytes include sheep, bovines, ovines, pigs, horses, rabbits, goats, guinea pigs, mice, 26 hamsters, rats, primates, humans, etc. In the preferred embodiments, the oocytes WO 99/45100 PCT/US99/04608 19- 1 will be obtained from primates or ungulates, a bovine.

2 Methods for isolation of oocytes are well known in the art. Essentially, 3 this will comprise isolating oocytes from the ovaries or reproductive tract of a 4 mammal or amphibian, a bovine. A readily available source of bovine oocytes is slaughterhouse materials.

6 For the successful use of techniques such as genetic engineering, nuclear 7 transfer and cloning, oocytes must generally be matured in vitro before these 8 cells may be used as recipient cells for nuclear transfer, and before they can be 9 fertilized by the sperm cell to develop into an embryo. This process generally requires collecting immature (prophase I) oocytes from animal ovaries, e.g., 11 bovine ovaries obtained at a slaughterhouse and maturing the oocytes in a 12 maturation medium prior to fertilization or enucleation until the oocyte attains 13 the metaphase II stage, which in the case of bovine oocytes generally occurs 14 about 18-24 hours post-aspiration. For purposes of the present invention, this period of time is known as the "maturation period." As used herein for 16 calculation of time periods, "aspiration" refers to aspiration of the immature 17 oocyte from ovarian follicles.

18 Additionally, metaphase II stage oocytes, which have been matured in 19 vivo have been successfully used in nuclear transfer techniques. Essentially, mature metaphase II oocytes are collected surgically from either non-superovu- 21 lated or superovulated cows or heifers 35 to 48 hours past the onset of estrus or 22 past the injection of human chorionic gonadotropin (hCG) or similar hormone.

23 The stage of maturation of the oocyte at enucleation and nuclear transfer 24 has been reported to be significant to the success of NT methods. (See e.g., Prather et al., Differentiation, 48, 1-8, 1991). In general, previous successful 26 mammalian embryo cloning practices use the metaphase II stage oocyte as the WO 99/45100 PCT/US99/04608 1 recipient oocyte because at this stage it is believed that the oocyte can be or is 2 sufficiently "activated" to treat the introduced nucleus as it does a fertilizing 3 sperm. In domestic animals, and especially cattle, the oocyte activation period 4 generally ranges from about 16-52 hours, preferably about 28-42 hours postaspiration.

6 For example, immature oocytes may be washed in HEPES buffered 7 hamster embryo culture medium (HECM) as described in Seshagine et al., Biol.

8 Reprod., 40, 544-606, 1989, and then placed into drops of maturation medium 9 consisting of 50 microliters of tissue culture medium (TCM) 199 containing fetal calf serum which contains appropriate gonadotropins such as luteinizing 11 hormone (LH) and follicle stimulating hormone (FSH), and estradiol under a 12 layer of lightweight paraffin or silicon at 39 0

C.

13 After a fixed time maturation period, which typically will range from 14 about 10 to 40 hours, and preferably about 16-18 hours, the oocytes will be enucleated. Prior to enucleation the oocytes will preferably be removed and 16 placed in HECM containing 1 milligram per milliliter of hyaluronidase prior to 17 removal of cumulus cells. This may be effected by repeated pipetting through 18 very fine bore pipettes or by vortexing briefly. The stripped oocytes are then 19 screened for polar bodies, and the selected metaphase II oocytes, as determined by the presence of polar bodies, are then used for nuclear transfer. Enucleation 21 follows.

22 Enucleation may be effected by known methods, such as described in U.S.

23 Patent No. 4,994,384 which is incorporated by reference herein. For example, 24 metaphase II oocytes are either placed in HECM, optionally containing micrograms per milliliter cytochalasin B, for immediate enucleation, or may be 26 placed in a suitable medium, for example CR1 aa, plus 10% estrus cow serum, WO 99/45100 PCT/US99/04608 -21 1 and then enucleated later, preferably not more than 24 hours later, and more 2 preferably 16-18 hours later.

3 Enucleation may be accomplished microsurgically using a micropipette 4 to remove the polar body and the adjacent cytoplasm. The oocytes may then be screened to identify those of which have been successfully enucleated. This 6 screening may be effected by staining the oocytes with 1 microgram per milliliter 7 33342 Hoechst dye in HECM, and then viewing the oocytes under ultraviolet 8 irradiation for less than 10 seconds. The oocytes that have been successfully 9 enucleated can then be placed in a suitable culture medium.

In the present invention, the recipient oocytes will preferably be 11 enucleated at a time ranging from about 10 hours to about 40 hours after the 12 initiation of in vitro maturation, more preferably from about 16 hours to about 13 24 hours after initiation of in vitro maturation, and most preferably about 16-18 14 hours after initiation of in vitro maturation.

A single animal or human cell or nucleus derived therefrom which is 16 typically heterologous to the enucleated oocyte will then be transferred into the 17 perivitelline space of the enucleated oocyte used to produce the NT unit. The 18 animal or human cell or nucleus and the enucleated oocyte will be used to 19 produce NT units according to methods known in the art. For example, the cells may be fused by electrofusion. Electrofusion is accomplished by providing a 21 pulse of electricity that is sufficient to cause a transient break down of the plasma 22 membrane. This breakdown of the plasma membrane is very short because the 23 membrane reforms rapidly. Essentially, if two adjacent membranes are induced 24 to break down, upon reformation the lipid bilayers intermingle and small channels will open between the two cells. Due to the thermodynamic instability of 26 such a small opening, it enlarges until the two cells become one. Reference is .WO 99/45100 PCT/US99/04608 -22- 1 made to U.S. Patent 4,997,384 by Prather et al., (incorporated by reference in its 2 entirety herein) for a further discussion of this process. A variety of electro- 3 fusion media can be used including sucrose, mannitol, sorbitol and phos- 4 phate buffered solution. Fusion can also be accomplished using Sendai virus as a fusogenic agent (Graham, Wister Inot. Symp. Monogr., 9, 19, 1969).

6 Also, in some cases with small donor nuclei) it may be preferable to 7 inject the nucleus directly into the oocyte rather than using electroporation 8 fusion. Such techniques are disclosed in Collas and Barnes, Mol. Reprod. Dev., 9 38:264-267 (1994), and incorporated by reference in its entirety herein.

Preferably, the human or animal cell and oocyte are electrofused in a 500 11 gm chamber by application of an electrical pulse of 90-120V for about 15 1sec, 12 about 24 hours after initiation of oocyte maturation. After fusion, the resultant 13 fused NT units are then placed in a suitable medium until activation, one 14 identified infra. Typically activation will be effected shortly thereafter, typically less than 24 hours later, and preferably about 4-9 hours later.

16 The NT unit may be activated by known methods. Such methods include, 17 culturing the NT unit at sub-physiological temperature, in essence by 18 applying a cold, or actually cool temperature shock to the NT unit. This may be 19 most conveniently done by culturing the NT unit at room temperature, which is cold relative to the physiological temperature conditions to which embryos are 21 normally exposed.

22 Alternatively, activation may be achieved by application of known 23 activation agents. For example, penetration of oocytes by sperm during 24 fertilization has been shown to activate prefusion oocytes to yield greater numbers of viable pregnancies and multiple genetically identical calves after 26 nuclear transfer. Also, treatments such as electrical and chemical shock or WO 99/45100 PCT/US99/04608 -23- 1 cycloheximide treatment may also be used to activate NT embryos after fusion.

2 Suitable oocyte activation methods are the subject of U.S. Patent No. 5,496,720, 3 to Susko-Parrish et al., which is herein incorporated by reference.

4 For example, oocyte activation may be effected by simultaneously or sequentially: 6 increasing levels of divalent cations in the oocyte, and 7 (ii) reducing phosphorylation of cellular proteins in the oocyte.

8 This will generally be effected by introducing divalent cations into the 9 oocyte cytoplasm, magnesium, strontium, barium or calcium, in the form of an ionophore. Other methods of increasing divalent cation levels include 11 the use of electric shock, treatment with ethanol and treatment with caged 12 chelators.

13 Phosphorylation may be reduced by known methods, by the addition 14 ofkinase inhibitors, serine-threonine kinase inhibitors, such as 6-dimethylamino-purine, staurosporine, 2-aminopurine, and sphingosine.

16 Alternatively, phosphorylation of cellular proteins may be inhibited by 17 introduction of a phosphatase into the oocyte, phosphatase 2A and 18 phosphatase 2B.

19 Activated NT units may be cultured in a suitable in vitro culture medium until the generation of embryonic or stem-like cells and cell colonies. Culture 21 media suitable for culturing and maturation of embryos are well known in the art.

22 Examples of known media, which may be used for bovine embryo culture and 23 maintenance, include Ham's F-10 10% fetal calf serum (FCS), Tissue Culture 24 Medium-199 (TCM-199) 10% fetal calf serum, Tyrodes-Albumin-Lactate- Pyruvate (TALP), Dulbecco's Phosphate Buffered Saline (PBS), Eagle's and 26 Whitten's media. One of the most common media used for the collection and WO 99/45100 PCT/US99/04608 -24- 1 maturation of oocytes is TCM-199, and 1 to 20% serum supplement including 2 fetal calf serum, newborn serum, estrual cow serum, lamb serum or steer serum.

3 A preferred maintenance medium includes TCM-199 with Earl salts, 10% fetal 4 calf serum, 0.2 MM Ma pyruvate and 50 gg/ml gentamicin sulphate. Any of the above may also involve co-culture with a variety of cell types such as granulosa 6 cells, oviduct cells, BRL cells and uterine cells and STO cells.

7 In particular, human epithelial cells of the endometrium secrete leukemia 8 inhibitory factor (LIF) during the preimplantation and implantation period.

9 Therefore, the addition of LIF to the culture medium could be of importance in enhancing the in vitro development of the reconstructed embryos. The use of 11 LIF for embronic or stem-like cell cultures has been described in U.S. Patent 12 5,712,156, which is herein incorporated by reference.

13 Another maintenance medium is described in U.S. Patent 5,096,822 to 14 Rosenkrans, Jr. et al., which is incorporated herein by reference. This embryo medium, named CR1, contains the nutritional substances necessary to support an 16 embryo. CR1 contains hemicalcium L-lactate in amounts ranging from 1.0 mM 17 to 10 mM, preferably 1.0 mM to 5.0 mM. Hemicalcium L-lactate is L-lactate 18 with a hemicalcium salt incorporated thereon.

19 Also, suitable culture medium for maintaining human embryonic cells in culture as discussed in Thomson et al., Science 282:1145-1147 (1998) and Proc.

21 Natl. Acad. Sci., USA 92:7844-7848 (1995).

22 Afterward, the cultured NT unit or units are preferably washed and then 23 placed in a suitable media, CRIaa medium, Ham's F-10, Tissue Culture 24 Media -199 (TCM-199). Tyrodes-Albumin-Lactate-Pyrovate (TALP) Dulbecco's Phosphate Buffered Saline (PBS), Eagle's or Whitten's, preferably containing 26 about 10% FCS. Such culturing will preferably be effected in well plates which WO 99/45100 PCT/US99/04608 1 contain a suitable confluent feeder layer. Suitable feeder layers include, by way 2 of example, fibroblasts and epithelial cells, fibroblasts and uterine epithelial 3 cells derived from ungulates, chicken fibroblasts, murine mouse or rat) 4 fibroblasts, STO and SI-m220 feeder cell lines, and BRL cells.

In the preferred embodiment, the feeder cells will comprise mouse 6 embryonic fibroblasts. Means for preparation of a suitable fibroblast feeder layer 7 are described in the example which follows and is well within the skill of the 8 ordinary artisan.

9 The NT units are cultured on the feeder layer until the NT units reach a size suitable for obtaining cells which may be used to produce embryonic stem- 11 like cells or cell colonies. Preferably, these NT units will be cultured until they 12 reach a size of at least about 2 to 400 cells, more preferably about 4 to 128 cells, 13 and most preferably at least about 50 cells. The culturing will be effected under 14 suitable conditions, about 38.5 C and 5% C0 2 with the culture medium changed in order to optimize growth typically about every 2-5 days, preferably 16 about every 3 days.

17 In the case of human cell/enucleated bovine oocyte derived NT units, 18 sufficient cells to produce an ES cell colony, typically on the order of about 19 cells, will be obtained about 12 days after initiation of oocyte activation.

However, this may vary dependent upon the particular cell used as the nuclear 21 donor, the species of the particular oocyte, and culturing conditions. One skilled 22 in the art can readily ascertain visually when a desired sufficient number of cells 23 has been obtained based on the morphology of the cultured NT units.

24 In the case of human/human nuclear transfer embryos, it may be advantageous to use culture medium known to be useful for maintaining human 26 cells in tissue culture. Examples of a culture media suitable for human embryo WO 99/45100 PCT/US99/04608 -26- 1 culture include the medium reported in Jones et al, Human Reprod., 13(1): 169- 2 177 (1998), the P 1-catalog #99242 medium, and the P- I catalog #99292 medium, 3 both available from Irvine Scientific, Santa Ana, California, and those used by 4 Thomson et al. (1998) and (1995). As discussed above, the cells used in the present invention will preferably 6 comprise mammalian somatic cells, most preferably cells derived from an 7 actively proliferating (non-quiescent) mammalian cell culture. In an especially 8 preferred embodiment, the donor cell will be genetically modified by the 9 addition, deletion or substitution of a desired DNA sequence. For example, the donor cell, a keratinocyte or fibroblast, of human, primate or bovine 11 origin, may be transfected or transformed with a DNA construct that provides for 12 the expression of a desired gene product, therapeutic polypeptide. Examples 13 thereof include lympokines, IGF-I, IGF-II, interferons, colony stimulating 14 factors, connective tissue polypeptides such as collagens, genetic factors, clotting factors, enzymes, enzyme inhibitors, etc.

16 Also, as discussed above, the donor cells may be modified prior to nuclear 17 transfer to achieve other desired effects, impaired cell lineage development, 18 enhanced embryonic development and/or inhibition of apoptosis. Examples of 19 desirable modifications are discussed further below.

One aspect of the invention will involve genetic modification of the donor 21 cell, a human cell, such that it is lineage deficient and therefore when used 22 for nuclear transfer it will be unable to give rise to a viable offspring. This is 23 desirable especially in the context of human nuclear transfer embryos, wherein 24 for ethical reasons, production of a viable embryo may be an unwanted outcome.

This can be effected by genetically engineering a human cell such that it is 26 incapable of differentiating into specific cell lineages when used for nuclear WO 99/45100 PCT/US99/04608 -27- 1 transfer. In particular, cells may be genetically modified such that when used as 2 nuclear transfer donors the resultant "embryos" do not contain or substantially 3 lack at least one of mesoderm, endoderm or ectoderm tissue.

4 It is anticipated that this can be accomplished by knocking out or impairing the expression of one or more mesoderm, endoderm or ectoderm 6 specific genes. Examples thereof include: 7 Mesdoerm: SRF, MESP-1, HNF-4, beta-I integrin, MSD; 8 Endoderm: GATA-6, GATA-4; 9 Ectoderm: RNA helicase A, H beta 58.

The above list is intended to be exemplary and non-exhaustive of known 11 genes which are involved in the development of mesoderm, endoderm and 12 ectoderm. The generation of mesoderm deficient, endoderm deficient and 13 ectoderm deficient cells and embryos has been previously reported in the 14 literature. See, Arsenian et al, EMBOJ., Vol. 17(2):6289-6299 (1998); Saga Y, Mech. Dev., Vol. 75(1-2):53-66 (1998); Holdener et al, Development, Voll 16 120(5):1355-1346 (1994); Chen et al, Genes Dev. Vol. 8(20):2466-2477 (1994); 17 Rohwedel et al, Dev. Biol., 201(2):167-189 (1998) (mesoderm); Morrisey et al, 18 Genes, Dev., Vol. 12(22):3579-3590 (1998); Soudais et al, Development, Vol.

19 121(11):3877-3888 (1995) (endoderm); and Lee et al, Proc. Natl. Acad. Sci.

USA, Vol. 95:(23):13709-13713 (1998); and Radice et al, Development, Vol.

21 111(3):801-811 (1991) (ectoderm).

22 In general, a desired somatic cell, a human keratinocyte, epithelial 23 cell or fibroblast, will be genetically engineered such that one or more genes 24 specific to particular cell lineages are "knocked out" and/or the expression of such genes significantly impaired. This may be effected by known methods, e.g., 26 homologous recombination. A preferred genetic system for effecting "knock- WO 99/45100 PCT/US99/04608 -28- 1 out" of desired genes is disclosed by Capecchi et al, U.S. Patents 5,631,153 and 2 5,464,764, which reports positive-negative selection (PNS) vectors that enable 3 targeted modification of DNA sequences in a desired mammalian genome. Such 4 genetic modification will result in a cell that is incapable of differentiating into a particular cell lineage when used as a nuclear transfer donor.

6 This genetically modified cell will be used to produce a lineage-defective 7 nuclear transfer embryo, that does not develop at least one of a functional 8 mesoderm, endoderm or ectoderm. Thereby, the resultant embryos, even if 9 implanted, into a human uterus, would not give rise to a viable offspring.

However, the ES cells that result from such nuclear transfer will still be useful 11 in that they will produce cells of the one or two remaining non-impaired lineage.

12 For example, an ectoderm deficient human nuclear transfer embryo will still give 13 rise to mesoderm and endoderm derived differentiated cells. An ectoderm 14 deficient cell can be produced by deletion and/or impairment of one or both of RNA helicase A or H beta 58 genes.

16 These lineage deficient donor cells may also be genetically modified to 17 express another desired DNA sequence.

18 Thus, the genetically modified donor cell will give rise to a lineage- 19 deficient blastocyst which, when plated, will differentiate into at most two of the embryonic germ layers.

21 Alternatively, the donor cell can be modified such that it is "mortal". This 22 can be achieved by expressing anti-sense or ribozyme telomerase genes. This 23 can be effected by known genetic methods that will provide for expression of 24 antisense DNA or ribozymes, or by gene knockout. These "mortal" cells, when used for nuclear transfer, will not be capable of differentiating into viable 26 offspring.

WO 99/45100 PCT/US99/04608 -29- 1 Another preferred embodiment of the present invention is the production 2 of nuclear transfer embryos that grow more efficiently in tissue culture. This is 3 advantageous in that it should reduce the requisite time and necessary fusions to 4 produce ES cells and/or offspring (if the blastocysts are to be implanted into a female surrogate). This is desirable also because it has been observed that 6 blastocysts and ES cells resulting from nuclear transfer may have impaired 7 development potential. While these problems may often be alleviated by 8 alteration of tissue culture conditions, an alternative solution is to enhance 9 embryonic development by enhancing expression of genes involved in embryonic development.

11 For example, it has been reported that the gene products of the Ped type, 12 which are members of the MHC I family, are of significant importance to 13 embryonic development. More specifically, it has been reported in the case of 14 mouse preimplantation embryos that the Q7 and Q9 genes are responsible for the "fast growth" phenotype. Therefore, it is anticipated that introduction of DNAs 16 that provide for the expression of these and related genes, or their human or other 17 mammalian counterparts into donor cells, will give rise to nuclear transfer 18 embryos that grow more quickly. This is particularly desirable in the context of 19 cross-species nuclear transfer embryos which may develop less efficiently in tissue culture than nuclear transfer embryos produced by fusion of cells or nuclei 21 of the same species.

22 In particular, a DNA construct containing the Q7 and/or Q9 gene will be 23 introduced into donor somatic cells prior to nuclear transfer. For example, an 24 expression construct can be constructed containing a strong constitutive mammalian promoter operably linked to the Q7 and/or Q9 genes, an IRES, one 26 or more suitable selectable markers, neomycin, ADA, DHFR, and a poly-A WO 99/45100 PCT/US99/04608 1 sequence, bGH polyA sequence. Also, it may be advantageous to further 2 enhance Q7 and Q9 gene expression by the inclusion of insulates. It is 3 anticipated that these genes will be expressed early on in blastocyst development 4 as these genes are highly conserved in different species, bovines, goats, porcine, dogs, cats, and humans. Also, it is anticipated that donor cells can be 6 engineered to affect other genes that enhance embryonic development. Thus, 7 these genetically modified donor cells should produce blastocysts and 8 preimplantation stage embryos more efficiently.

9 Still another aspect of the invention involves the construction of donor cells that are resistant to apoptosis, programmed cell death. It has been 11 reported in the literature that cell death related genes are present in 12 preimplantation stage embryos. (Adams et al, Science, 281(5381):1322-1326 13 (1998)). Genes reported to induce apoptosis include, Bad, Bok, BH3, Bik, 14 Hrk, BNIP3, BimL, Bad, Bid, and EGL-1. By contrast, genes that reportedly protect cells from programmed cell death include, by way of example, BcL-XL, 16 Bcl-w, Mcl-1, Al, Nr-13, BHRF-1, LMW5-HL, ORF16, Ks-Bel-2, E1B-19K, 17 and CED-9.

18 Thus, donor cells can be constructed wherein genes that induce apoptosis 19 are "knocked out" or wherein the expression of genes that protect the cells from apoptosis is enhanced or turned on during embryonic development.

21 For example, this can be effected by introducing a DNA construct that 22 provides for regulated expression of such protective genes, Bcl-2 or related 23 genes during embryonic development. Thereby, the gene can be "turned on" by 24 culturing the embryo under specific growth conditions. Alternatively, it can be linked to a constitutive promoter.

26 More specifically, a DNA construct containing a Bcl-2 gene operably WO 99/45100 PCT/US99/04608 -31 1 linked to a regulatable or constitutive promoter, PGK, SV40, CMV, 2 ubiquitin, or beta-actin, an IRES, a suitable selectable marker, and a poly-A 3 sequence can be constructed and introduced into a desired donor mammalian 4 cell, human keratinocyte or fibroblast.

These donor cells, when used to produce nuclear transfer embryos, should 6 be resistant to apoptosis and thereby differentiate more efficiently in tissue 7 culture. Thereby, the speed and/or number of suitable preimplantation embryos 8 produced by nuclear transfer can be increased.

9 Another means of accomplishing the same result is to impair the expression of one or more genes that induce apoptosis. This will be effected by 11 knock-out or by the use of antisense or ribozymes against genes that are 12 expressed in and which induce apoptosis early on in embryonic development.

13 Examples thereof are identified above. Still alternatively, donor cells may be 14 constructed containing both modifications, impairment of apoptosis-inducing genes and enhanced expression of genes that impede or prevent apoptosis. The 16 construction and selection of genes that affect apoptosis, and cell lines that 17 express such genes, is disclosed in U.S. Patent No. 5,646,008, Craig B.

18 Thompson et al inventors, and assigned to the University of Michigan. This 19 patent is incorporated by reference herein.

2 0 One means of enhancing efficiency is to select cells of a particular cell 21 cycle stage as the donor cell. It has been reported that this can have significant 22 effects on nuclear transfer efficiency. (Bares et al, Mol. Reprod. Devel., 23 36(1):33-41 (1993). Different methods for selecting cells of a particular cell 24 cycle stage have been reported and include serum starvation (Campbell et al, Nature, 380:64-66 (1996); Wilmut et al, Nature, 385:810-813 (1997), and 26 chemical synchronization (Urbani et al, Exp. Cell Res., 219(1):159-168 (1995).

WO 99/45100 PCT/US99/04608 -32- 1 For example, a particular cyclin DNA may be operably linked to a regulatory 2 sequence, together with a detectable marker, green fluorescent protein 3 (GFP), followed by the cyclin destruction box, and optionally insulation 4 sequences to enhance cyclin and marker protein expression. Thereby, cells of a desired cell cycle can be easily visually detected and selected for use as a nuclear 6 transfer donor. An example thereof is the cyclin D1 gene in order to select for 7 cells that are in G 1. However, any cyclin gene should be suitable for use in the 8 claimed invention. (See, King et al, Mol. Biol. Cell, Vol. 7(9):1343-1357 9 (1996)).

However, a less invasive or more efficient method for producing cells of 11 a desired cell cycle stage are needed. It is anticipated that this can be effected by 12 genetically modifying donor cells such that they express specific cyclins under 13 detectable conditions. Thereby, cells of a specific cell cycle can be readily 14 discerned from other cell cycles.

Cyclins are proteins that are expressed only during specific stages of the 16 cell cycle. They include cyclin Dl, D2 and D3 in G1 phase, cyclin B1 and B2 17 in G2/M phase and cyclin E, A and H in S phase. These proteins are easily 18 translated and destroyed in the cytogolcytosol. This "transient" expression of 19 such proteins is attributable in part to the presence of a "destruction box", which is a short amino acid sequence that is part of the protein that functions as a tag 21 to direct the prompt destruction of these proteins via the ugiquitin pathway.

22 (Adams et al, Science, 281 (5321):1322-1326 (1998)).

23 In the present invention, donor cells will be constructed that express one 24 or more of such cyclin genes under easily detectable conditions, preferably visualizable, by the use of a fluorescent label. For example, a particular 26 cyclin DNA may be operably linked to a regulatory sequence, together with a WO 99/45100 PCT/US99/04608 -33- 1 detectable marker, green fluorescent protein (GFP), followed by the cyclin 2 destruction box, and optionally insulation sequences to enhance cyclin and/or 3 marker protein expression. Thereby, cells of a desired cell cycle can be easily 4 visually detected and selected for use as a nuclear transfer donor. An example thereof is the cyclin D1 gene which can be used to select for cells that are in G1.

6 However, any cyclin gene should be suitable for use in the claimed invention.

7 (See, King et al, Mol. Biol. Cell, Vol. 7(9):1343-1357 (1996)).

8 Still another aspect of the invention is a method for enhancing nuclear 9 transfer efficiency, preferably in a cross-species nuclear transfer process. While the present inventors have demonstrated that nuclei or cells of one species when 11 inserted or fused with an enucleated oocyte of a different species can give rise 12 to nuclear transfer embryos that produce blastocysts, which embryos can give 13 rise to ES cell lines, the efficiency of such process is quite low. Therefore, many 14 fusions typically need to be effected to produce a blastocyst the cells of which may be cultured to produce ES cells and ES cell lines.

16 Yet another means for enhancing the development of nuclear transfer 17 embryos in vitro is by optimizing culture conditions. One means of achieving 18 this result will be to culture NT embryos under conditions impede apoptosis.

19 With respect to this embodiment of the invention, it has been found that proteases such as capsases can cause oocyte death by apoptosis similar to other 21 cell types. (See, Jurisicosva et al, Mol. Reprod. Devel., 51(3):243-253 (1998).) 22 It is anticipated that blastocyst development will be enhanced by including 23 in culture media used for nuclear transfer and to maintain blastocysts or culture 24 preimplantation stage embryos one or more capsase inhibitors. Such inhibitors include by way of example capsase-4 inhibitor I, capsase-3 inhibitor I, capsase-6 26 inhibitor II, capsase-9 inhibitor II, and capsase-1 inhibitor I. the amount thereof WO 99/45100 PCT/US99/04608 -34- 1 will be an amount effective to inhibit apoptosis, 0.00001 to 5.0% by weight 2 of medium; more preferably 0.01% to 1.0% by weight of medium. Thus, the 3 foregoing methods may be used to increase the efficiency of nuclear transfer by 4 enhancing subsequent blastocyst and embryo development in tissue culture.

After NT units of the desired size are obtained, the cells are mechanically 6 removed from the zone and are then used to produce embryonic or stem-like 7 cells and cell lines. This is preferably effected by taking the clump of cells s which comprise the NT unit, which typically will contain at least about 50 cells, 9 washing such cells, and plating the cells onto a feeder layer, irradiated fibroblast cells. Typically, the cells used to obtain the stem-like cells or cell colonies 11 will be obtained from the inner most portion of the cultured NT unit which is 12 preferably at least 50 cells in size. However, NT units of smaller or greater cell 13 numbers as well as cells from other portions of the NT unit may also be used to 14 obtain ES-like cells and cell colonies.

It may be that a longer exposure of donor cell DNA to the oocyte's 16 cytosol would facilitate the dedifferentiation process. In this regard, recloning 17 could be accomplished by taking blastomeres from a reconstructed embryo and 18 fusing them with a new enucleated oocyte. Alternatively, the donor cell may be 19 fused with an enucleated oocyte and 4 to 6 hours later, without activation, chromosomes may be removed and fused with a younger oocyte. Activation 21 would occur thereafter.

22 The cells are maintained in the feeder layer in a suitable growth medium, 23 alpha MEM supplemented with 10% FCS and 0.1 mM beta- 24 mercaptoethanol (Sigma) and L-glutamine. The growth medium is changed as often as necessary to optimize growth, about every 2-3 days.

26 This culturing process results in the formation of embryonic or stem-like .WO 99/45100 PCT/US99/04608 1 cells or cell lines. In the case of human cell/bovine oocyte derived NT embryos, 2 colonies are observed by about the second day of culturing in the alpha MEM 3 medium. However, this time may vary dependent upon the particular nuclear 4 donor cell, specific oocyte and culturing conditions. One skilled in the art can vary the culturing conditions as desired to. optimize growth of the particular 6 embryonic or stem-like cells.

7 The embryonic or stem-like cells and cell colonies obtained will typically 8 exhibit an appearance similar to embryonic or stem-like cells of the species used 9 as the nuclear cell donor rather than the species of the donor oocyte. For example, in the case of embryonic or stem-like cells obtained by the transfer of 11 a human nuclear donor cell into an enucleated bovine oocyte, the cells exhibit a 12 morphology more similar to mouse embryonic stem cells than bovine ES-like 13 cells.

14 More specifically, the individual cells of the human ES-line cell colony are not well defined, and the perimeter of the colony is refractive and smooth in 16 appearance. Further, the cell colony has a longer cell doubling time, about twice 17 that of mouse ES cells. Also, unlike bovine and porcine derived ES cells, the 18 colony does not possess an epithelial-like appearance.

19 As discussed above, it has been reported by Thomson, in U.S. Patent 5,843,780, that primate stem cells are SSEA-1 SSEA-4 TRA-1-60 21 TRA-1-81 and alkaline phosphatase It is anticipated that human and 22 primate ES cells produced according to the present methods will exhibit similar 23 or identical marker expression.

24 Alternatively, that such cells are actual human or primate embryonic stem cells will be confirmed based on their capability of giving rise to all of 26 mesoderm, ectoderm and endoderm tissues. This will be demonstrated by WO 99/45100 PCT/US99/04608 -36- 1 culturing ES cells produced according to the invention under appropriate 2 conditions, as disclosed by Thomsen, U.S. Patent 5,843,780, incorporated 3 by reference in its entirety herein.

4 The resultant embryonic or stem-like cells and cell lines, preferably human embryonic or stem-like cells and cell lines, have numerous therapeutic 6 and diagnostic applications. Most especially, such embryonic or stem-like cells 7 may be used for cell transplantation therapies. Human embryonic or stem-like 8 cells have application in the treatment of numerous disease conditions.

9 In this regard, it is known that mouse embryonic stem (ES) cells are capable of differentiating into almost any cell type, hematopoietic stem 11 cells. Therefore, human embryonic or stem-like cells produced according to the 12 invention should possess similar differentiation capacity. The embryonic or 13 stem-like cells according to the invention will be induced to differentiate to 14 obtain the desired cell types according to known methods. For example, the subject human embryonic or stem-like cells may be induced to differentiate into 16 hematopoietic stem cells, muscle cells, cardiac muscle cells, liver cells, cartilage 17 cells, epithelial cells, urinary tract cells, etc., by culturing such cells in 18 differentiation medium and under conditions which provide for cell differentia- 19 tion. Medium and methods which result in the differentiation of embryonic stem cells are known in the art as are suitable culturing conditions.

21 For example, Palacios et al, Proc. Natl. Acad. Sci., USA, 92:7530-7537 22 (1995) teaches the production of hematopoietic stem cells from an embryonic 23 cell line by subjecting stem cells to an induction procedure comprising initially 24 culturing aggregates of such cells in a suspension culture medium lacking retinoic acid followed by culturing in the same medium containing retinoic acid, 26 followed by transferral of cell aggregates to a substrate which provides for cell WO 99/45100 PCT/US99/04608 -37- 1 attachment.

2 Moreover, Pedersen, J. Reprod. Fertil. Dev., 6:543-552 (1994) is a review 3 article which references numerous articles disclosing methods for in vitro 4 differentiation of embryonic stem cells to produce various differentiated cell types including hematopoietic cells, muscle, cardiac muscle, nerve cells, among 6 others.

7 Further, Bain et al, Dev. Biol., 168:342-357 (1995) teaches in vitro 8 differentiation of embryonic stem cells to produce neural cells which possess 9 neuronal properties. These references are exemplary of reported methods for obtaining differentiated cells from embryonic or stem-like cells. These 11 references and in particular the disclosures therein relating to methods for 12 differentiating embryonic stem cells are incorporated by reference in their 13 entirety herein.

14 Thus, using known methods and culture medium, one skilled in the art may culture the subject embryonic or stem-like cells to obtain desired 16 differentiated cell types, neural cells, muscle cells, hematopoietic cells, etc.

17 In addition, the use of inducible Bcl-2 or Bcl-xl might be useful for 18 enhancing in vitro development of specific cell lineages. In vivo, Bcl-2 prevents 19 many, but not all, forms of apoptotic cell death that occur during lymphoid and neural development. A thorough discussion of how Bcl-2 expression might be 21 used to inhibit apoptosis of relevent cell lineages following transfection of donor 22 cells is disclosed in U.S. Patent No. 5,646,008, which is herein incorporated by 23 reference.

24 The subject embryonic or stem-like cells may be used to obtain any desired differentiated cell type. Therapeutic usages of such differentiated human 26 cells are unparalleled. For example, human hematopoietic stem cells may be WO 99/45100 PCT/US99/04608 -38- 1 used in medical treatments requiring bone marrow transplantation. Such 2 procedures are used to treat many diseases, late stage cancers such as 3 ovarian cancer and leukemia, as well as diseases that compromise the immune 4 system, such as AIDS. Hematopoietic stem cells can be obtained, by fusing adult somatic cells of a cancer or AIDS patient, epithelial cells or lympho- 6 cytes with an enucleated oocyte, bovine oocyte, obtaining embryonic or 7 stem-like cells as described above, and culturing such cells under conditions 8 which favor differentiation, until hematopoietic stem cells are obtained. Such 9 hematopoietic cells may be used in the treatment of diseases including cancer and AIDS.

11 Alternatively, adult somatic cells from a patient with a neurological 12 disorder may be fused with an enucleated animal oocyte, a primate or 13 bovine oocyte, human embryonic or stem-like cells obtained therefrom, and such 14 cells cultured under differentiation conditions to produce neural cell lines.

Specific diseases treatable by transplantation of such human neural cells include, 16 by way of example, Parkinson's disease, Alzheimer's disease, ALS and cerebral 17 palsy, among others. In the specific case of Parkinson's disease, it has been 18 demonstrated that transplanted fetal brain neural cells make the proper connec- 19 tions with surrounding cells and produce dopamine. This can result in long-term reversal of Parkinson's disease symptoms.

21 To allow for specific selection of differentiated cells, donor cells may be 22 transfected with selectable markers expressed via inducible promoters, thereby 23 permitting selection or enrichment of particular cell lineages when 24 differentiation is induced. For example, CD34-neo may be used for selection of hematopoietic cells, Pwl-neo for muscle cells, Mash-l-neo for sympathetic 26 neurons, Mal-neo for human CNS heurons of the grey matter of the cerebral WO 99/45100 PCT/US99/04608 -39- 1 cortex, etc.

2 The great advantage of the subject invention is that it provides an 3 essentially limitless supply of isogenic or synegenic human cells suitable for 4 transplantation. Therefore, it will obviate the significant problem associated with current transplantation methods, rejection of the transplanted tissue which 6 may occur because of host-vs-graft or graft-vs-host rejection. Conventionally, 7 rejection is prevented or reduced by the administration of anti-rejection drugs 8 such as cyclosporine. However, such drugs have significant adverse side-effects, 9 immunosuppression, carcinogenic properties, as well as being very expensive. The present invention should eliminate, or at least greatly reduce, the 11 need for anti-rejection drugs.

12 Other diseases and conditions treatable by isogenic cell therapy include, 13 by way of example, spinal cord injuries, multiple sclerosis, muscular dystrophy, 14 diabetes, liver diseases, hypercholesterolemia, heart diseases, cartilage replacement, bums, foot ulcers, gastrointestinal diseases, vascular diseases, 16 kidney disease, urinary tract disease, and aging related diseases and conditions.

17 Also, human embryonic or stem-like cells produced according to the 18 invention may be used to produce genetically engineered or transgenic human 19 differentiated cells. Essentially, this will be effected by introducing a desired gene or genes, which may be heterologous, or removing all or part of an 21 endogenous gene or genes of human embryonic or stem-like cells produced 22 according to the invention, and allowing such cells to differentiate into the de- 23 sired cell type. A preferred method for achieving such modification is by 24 homologous recombination because such technique can be used to insert, delete or modify a gene or genes at a specific cite or cites in the stem-like cell genome.

26 This methodology can be used to replace defective genes, defective WO 99/45100 PCT/US99/04608 1 immune system genes, cystic fibrosis genes, or to introduce genes which result 2 in the expression of therapeutically beneficial proteins such as growth factors, 3 lymphokines, cytokines, enzymes, etc. For example, the gene encoding brain 4 derived growth factor may be introduced into human embryonic or stem-like cells, the cells differentiated into neural cells and the cells transplanted into a 6 Parkinson's patient to retard the loss of neural cells during such disease.

7 Previously, cell types transfected with BDNF varied from primary cells 8 to immortalized cell lines, either neural or non-neural (myoblast and fibroblast) 9 derived cells. For example, astrocytes have been transfected with BDNF gene using retroviral vectors, and the cells grafted into a rat model of Parkinson's 11 disease (Yoshimoto et al., Brain Research, 691:25-36, (1995)).

12 This ex vivo therapy reduced Parkinson's-like symptoms in the rats up to 13 45% 32 days after transfer. Also, the tyrosine hydroxylase gene has been placed 14 into astrocytes with similar results (Lundberg et al., Develop. Neurol., 139:39-53 (1996) and references cited therein).

16 However, such ex vivo systems have problems. In particular, retroviral 17 vectors currently used are down-regulated in vivo and the transgene is only 18 transiently expressed (review by Mulligan, Science, 260:926-932 (1993)). Also, 19 such studies used primary cells, astrocytes, which have finite life span and replicate slowly. Such properties adversely affect the rate of transfection and 21 impede selection of stably transfected cells. Moreover, it is almost impossible 22 to propagate a large population of gene targeted primary cells to be used in 23 homologous recombination techniques.

24 By contrast, the difficulties associated with retroviral systems should be eliminated by the use of human embryonic or stem-like cells. It has been demon- 26 strated previously by the subject assignee that cattle and pig embryonic cell lines WO 99/45100 PCT/US99/04608 -41- 1 can be transfected and selected for stable integration ofheterologous DNA. Such 2 methods are described in commonly assigned U.S. Serial No. 08/626,054, filed 3 April 1, 1996, incorporated by reference in its entirety. Therefore, using such 4 methods or other known methods, desired genes may be introduced into the subject human embryonic or stem-like cells, and the cells differentiated into desired 6 cell types, hematopoietic cells, neural cells, pancreatic cells, cartilage cells, 7 etc.

8 Genes which may be introduced into the subject embryonic or stem-like 9 cells include, by way of example, epidermal growth factor, basic fibroblast growth factor, glial derived neurotrophic growth factor, insulin-like growth 11 factor (I and II), neurotrophin-3, neurotrophin-4/5, ciliary neurotrophic factor, 12 AFT-1, cytokine genes (interleukins, interferons, colony stimulating factors, 13 tumor necrosis factors (alpha and beta), etc.), genes encoding therapeutic 14 enzymes, collagen, human serum albumin, etc.

In addition, it is also possible to use one of the negative selection systems 16 now known in the art for eliminating therapeutic cells from a patient if necessary.

17 For example, donor cells transfected with the thymidine kinase (TK) gene will 18 lead to the production of embryonic cells containing the TK gene.

19 Differentiation of these cells will lead to the isolation of therapeutic cells of interest which also express the TK gene. Such cells may be selectively 21 eliminated at any time from a patient upon gancyclovir administration. Such a 22 negative selection system is described in U.S. Patent No. 5,698,446, and is herein 23 incorporated by reference.

24 The subject embryonic or stem-like cells, preferably human cells, also may be used as an in vitro model of differentiation, in particular for the study of 26 genes which are involved in the regulation of early development.

WO 99/45100 PCT/US99/04608 -42- 1 Also, differentiated cell tissues and organs using the subject embryonic 2 or stem-like cells may be used in drug studies.

3 Further, the subject embryonic or stem-like cells may be used as nuclear 4 donors for the production of other embryonic or stem-like cells and cell colonies.

In order to more clearly describe the subject invention, the following 6 examples are provided.

7 EXAMPLE 1 8 MATERIALS AND METHODS 9 Donor Cells for Nuclear Transfer Epithelial cells were lightly scraped from the inside of the mouth of a 11 consenting adult with a standard glass slide. The cells were washed off the slide 12 into a petri dish containing phosphate buffered saline without Ca or Mg. The 13 cells were pipetted through a small-bore pipette to break up cell clumps into a 14 single cell suspension. The cells were then transferred into a microdrop of TL- HEPES medium containing 10% fetal calf serum (FCS) under oil for nuclear 16 transfer into enucleated cattle oocytes.

17 Nuclear Transfer Procedures 18 Basic nuclear transfer procedures have been described previously.

19 Briefly, after slaughterhouse oocytes were matured in vitro the oocytes were 2 0 stripped of cumulus cells and enucleated with a beveled micropipette at ap- 21 proximately 18 hours post maturation (hpm). Enucleation was confirmed in TL- 22 HEPES medium plus bisbenzimide (Hoechst 33342, 3 jig/ml; Sigma). Individual 23 donor cells were then placed into the perivitelline space of the recipient oocyte.

24 The bovine oocyte cytoplasm and the donor nucleus (NT unit) are fused together using electrofusion techniques. One fusion pulse consisting of 90 V for 15 psec 26 was applied to the NT unit. This occurred at 24 hours post-initiation of WO 99/45100 PCT/US99/04608 -43- 1 maturation (hpm) of the oocytes. The NT units were placed in CRlaa medium 2 until 28 hpm.

3 The procedure used to artificially activate oocytes has been described 4 elsewhere. NT unit activation was at 28 hpm. A brief description of the activation procedure is as follows: NT units were exposed for four min to 6 ionomycin (5 tM; CalBiochem, La Jolla, CA) in TL-HEPES supplemented with 7 1 mg/ml BSA and then washed for five min in TL-HEPES supplemented with 8 30 mg/ml BSA. The NT units were then transferred into a microdrop of CR1 aa 9 culture medium containing 0.2 mM DMAP (Sigma) and cultured at 38.5 °C

CO

2 for four to five hours. The NT units were washed and then placed in a 11 CRlaa medium plus 10% FCS and 6 mg/ml BSA in four well plates containing 12 a confluent feeder layer of mouse embryonic fibroblasts (described below). The 13 NT units were cultured for three more days at 38.5 C and 5% CO 2 The culture 14 medium was changed every three days until day 12 after the time of activation.

At this time NT units reaching the desired cell number, about 50 cell 16 number, were mechanically removed from the zona and used to produce 17 embryonic cell lines. A photograph of an NT unit obtained as described above 18 is contained in Figure 1.

19 Fibroblast feeder layer Primary cultures of embryonic fibroblasts were obtained from 14-16 day 21 old murine fetuses. After the head, liver, heart and alimentary tract were 22 aseptically removed, the embryos were minced and incubated for 30 minutes at 23 37 0 C in prewarmed trypsin EDTA solution (0.05% trypsin/0.02% EDTA; 24 GIBCO, Grand Island, NY). Fibroblast cells were plated in tissue culture flasks and cultured in alpha-MEM medium (BioWhittaker, Walkersville, MD) supple- 26 mented with 10% fetal calf serum (FCS) (Hyclone, Logen, UT), penicillin (100 WO 99/45100 PCT/US99/04608 -44- 1 IU/ml) and streptomycin (50 gl/ml). Three to four days after passage, embryonic 2 fibroblasts, in 35 x 10 Nunc culture dishes (Baxter Scientific, McGaw Park, IL), 3 were irradiated. The irradiated fibroblasts were grown and maintained in a 4 humidified atmosphere with 5% CO 2 in air at 37°C. The culture plates which had a uniform monolayer of cells were then used to culture embryonic cell lines.

6 Production of embryonic cell line.

7 NT unit cells obtained as described above were washed and plated directly 8 onto irradiated feeder fibroblast cells. These cells included those of the inner 9 portion of the NT unit. The cells were maintained in a growth medium consisting of alpha MEM supplemented with 10% FCS and 0.1 mM beta- 11 mercaptoethanol (Sigma). Growth medium was exchanged every two to three 12 days. The initial colony was observed by the second day of culture. The colony 13 was propagated and exhibits a similar morphology to previously disclosed mouse 14 embryonic stem (ES) cells. Individual cells within the colony are not well defined and the perimeter of the colony is refractile and smooth in appearance.

16 The cell colony appears to have a slower cell doubling time than mouse ES cells.

17 Also, unlike bovine and porcine derived ES cells, the colony does not have an 18 epithelial appearance thus far. Figures 2 through 5 are photographs of ES-like 19 cell colonies obtained as described, supra.

Production of Differentiated Human Cells 21 The human embryonic cells obtained are transferred to a differentiation 22 medium and cultured until differentiated human cell types are obtained.

23

RESULTS

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

WO 99/45100 PCT/US99/04608 1 TABLE 1 2 Cell type No. NT No. NT units No. NT units to 4 No. NT units to units made 2 cell stage 16 cell stage 16 400 cell stage 3 lymphocytes 18 12 3 0 4 oral cavity epithelium 34 18 3 1(3%) 6 adult fibro- 12 (4 cell; 26%) 7 blasts 46 4 8 (8-16 cells; 17.4%) 8 9 The one NT unit that developed a structure having greater than 16 cells 0 was plated down onto a fibroblast feeder layer. This structure was attached to 1 the feeder layer and started to propagate forming a colony with a ES cell-like 2 morphology (See, Figure Moreover, although the 4 to 16 cell stage 3 structures were not used to try and produce an ES cell colony, it has been previ- 4 ously shown that this stage is capable of producing ES or ES-like cell lines (mouse, Eistetter et al., Devel. Growth and Differ., 31:275-282 (1989); Bovine, 6 Stice et al., 1996)). Therefore, it is expected that 4 16 cell stage NT units 7 should also give rise to embryonic or stem-like cells and cell colonies.

8 Also, similar results were obtained upon fusion of an adult human 9 keratinocyte cell line with an enucleated bovine oocyte, which was cultured in 0 media comprising ACM, uridine, glucose, and 1000 IU of LIF. Out of 1 reconstructed embryos, 22 cleaved and one developed into a blastocyst at about 2 day 12. This blastocyst was plated and the production of an ES cell line is 3 ongoing.

4 While the present invention has been described and illustrated herein by reference to various specific materials, procedures, and examples, it is under- 6 stood that the invention is not restricted to the particular material, combinations -46- 1 of materials, and procedures selected for that purpose. Numerous variations of such details can be implied and will be appreciated by those skilled in the art.

"Comprises/comprising" when used in this specification is taken to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

*S*

C o o

Claims

1. A method of producing stem-like cells comprising the following steps: inserting a desired non-quiescent differentiated human or mammalian cell or cell nucleus into an enucleated animal oocyte, wherein such oocyte is derived from a different animal species than the human or mammalian cell under conditions suitable for the formation of a nuclear transfer (NT) unit; (ii) activating the resultant NT unit; (iii) culturing said activated NT unit until greater than the 2 cell developmental stage; and (iv) culturing cells obtained from said cultured NT unit to obtain stem- like cells, wherein said donor cell is genetically modified by the insertion, removal or modification of a desired gene.

2. The method of claim 1, wherein said donor cell is genetically modified to impair S the development of at least one of endoderm, mesoderm and ectoderm.

3. The method of claim 2, wherein said donor cell is genetically modified to impair the development of endoderm, and said genetic modification alters the expression of a gene selected from the group consisting of GATA 6 and GATA4. 48

4. The method of claim 2, wherein said donor cell is genetically modified to impair the development of mesoderm, and said genetic modification alters the expression of a gene selected from the group consisting of SRF, MESP 1, HNF4, beta-I integrin and MSD. The method of claim 2, wherein said donor cell is genetically modified to impair the development of ectoderm, and said genetic modification alters the expression of a gene selected from the group consisting of RNA helicase A and H beta 58.

6. The method of claim 1, wherein said donor cell is genetically modified to enhance the development of at least one tissue of endoderm, mesoderm or ectoderm.

7. The method of claim 6, wherein said donor cell is transfected with a selectable marker gene expressed via an inducible promoter to allow for selection or enrichment of a particular cell lineage. :Ii 8. The method of claim 6, wherein said selectable marker gene is neo.

9. The method of claim 1, wherein said donor cell is genetically modified to enhance embryonic development. The method of claim 9, wherein said donor cell is transfected with at least one gene selected from the group consisting of MHC genes, Ped genes, Q7 genes and Q9 genes. 49

11. The method of claim 1, wherein said donor cell is genetically modified to be resistant to apoptosis.

12. The method of claim 11, wherein said genetic modification results in the reduced expression or blocked expression of at least one gene that enhances apoptosis.

13. The method of claim 12, wherein said genetic modification is selected from the group consisting of a "knock out" of said gene and expression of an antisense or ribozyme RNA that targets said gene.

14. The method of claim 12, wherein said at least one gene is selected from the group consisting of Bad, Bok, BH3, Bik, Hrk, BNIP3, BIML, Bid and EGL-1.

15. The method of claim 11 wherein said genetic modification results in the activation of gene expression of at least one gene that protects cells against apoptosis.

16. The method of claim 15, wherein said at least one gene is selected from the group consisting of Bcl-XL, Bcl-w, Mcl-1, Al, Nrl3, BHRF1, LMW5HL, ORF16, KS-Bel-2, E1B- 19K and CED-9.

17. The method of claim 15, wherein said at least one gene is operably linked to a regulatable or constitutive promoter.

18. The method of claim 17, wherein said promoter is selected from the group consisting of PGK, SV40, CMV, ubiquitin and beta-actin.

19. The method of claim 1, wherein said donor cell is genetically modified to allow for negative selection. The method of claim 19, wherein said donor cell is genetically modified to express a gene encoding thymidine kinase (TK) or an antisense or ribozyme construct specific for the telomerase gene.

21. The method of claim 1, wherein said donor cell is genetically modified with a transfected gene expressing a therapeutic polypeptide.

22. The method of claim 21, wherein said therapeutic polypeptide is selected from the group consisting of lymphokines such as IGFI and IGFII, interferons, colony stimulating factors, connective tissue polypeptides such as collagens, genetic factors, clotting factors, enzymes and enzyme inhibitors.

23. An improved method of producing stem-like cells comprising the following steps: inserting a desired differentiated human or mammalian cell or cell nucleus into an enucleated animal oocyte, wherein such oocyte is derived from a different animal species than the human or mammalian cell under conditions suitable for the formation of a nuclear transfer (NT) unit; (ii) activating the resultant NT unit; (iii) culturing said activated NT unit until greater than the 2 cell developmental stage; and (iv) culturing cells obtained from said cultured NT unit to obtain stem- like cells, wherein said improvement comprises using donor cells of a specific cell cycle stage.

24. The method of claim 23, wherein said donor cells are non-quiescent. The method of claim 23, wherein said cell cycle stage is the G1 stage.

26. The method of claim 23, wherein said donor cells of a specific cell cycle stage are selected by transfecting suitable donor cells with a genetic construct comprising a cyclin gene operably linked to a detectable marker.

27. The method of claim 25, wherein said G 1 donor cells are selected by transfecting suitable donor cells with a genetic construct comprising a cyclin D1 gene operably linked to a detectable marker.

28. The method of claim 23, wherein said donor cells are arrested at mitosis prior to nuclear transfer.

29. The method of claim 28, wherein said cells are arrested at mitosis using cyclohexamide. An improved method of producing stem-like cells comprising the following steps: inserting a desired differentiated human or mammalian cell or cell nucleus into an enucleated animal oocyte, wherein such oocyte is derived from a different animal species than the human or mammalian cell under conditions suitable for the formation of a nuclear transfer (NT) unit; (ii) activating the resultant NT unit; (iii) culturing said activated NT unit until greater than the 2 cell developmental stage; and (iv) culturing cells obtained from said cultured NT unit to obtain stem- S like cells, wherein said improvement comprises inhibiting apoptosis of said cells obtained from said cultured NT unit by culturing said cells in the presence of protease inhibitors.

31. The method of claim 30, wherein said protease inhibitor is a caspase inhibitor selected from the group consisting of caspase 4-inhibitor I, caspase 3-inhibitor I, caspase 6- inhibitor II, caspase 9-inhibitor II and caspase 1-inhibitor I.

32. An improved method of producing stem-like cells comprising the following steps: inserting a desired differentiated human or mammalian cell or cell nucleus into an enucleated animal oocyte, wherein such oocyte is derived from a different animal species than the human or mammalian cell under conditions suitable for the formation of a nuclear transfer (NT) unit; (ii) activating the resultant NT unit; (iii) culturing said activated NT unit until greater than the 2 cell developmental stage; and (iv) culturing cells obtained from said cultured NT unit to obtain stem- like cells, wherein said improvement comprises exposing said donor cell or NT unit to an agent that enhances embryonic development.

33. The method of claim 32, wherein said agent inhibits histone deacetylase.

34. The method of claim 33, wherein said deacetylase inhibitor is selected from the group consisting of Trichostatin A and butyrate. The method of claim 33, wherein said agent increases access of transcription factors to DNA regulatory sequences.

36. The method of claim 35, wherein said agent is a DNA demethylating agent. 54

37. The method of claim 36, wherein said agent is

38. An improved method of producing stem-like cells comprising the following steps: inserting a desired differentiated human or mammalian cell or cell nucleus into an enucleated animal oocyte, wherein such oocyte is derived from a different animal species than the human or mammalian cell under conditions suitable for the formation of a nuclear transfer (NT) unit; 1 (ii) activating the resultant NT unit; e (iii) culturing said activated NT unit until greater than the 2 cell developmental stage; and (iv) culturing cells obtained from said cultured NT unit to obtain stem- like cells, i wherein said improvement comprises a recloning step whereby said donor DNA is provided with increased exposure to the cytosol of the recipient oocyte.

39. The method of claim 38, wherein said recloning step involves taking said NT unit or a cell or cells from said NT unit or the nuclei therefrom and fusing them with a new enucleated oocyte. The method of claim 38, wherein said recloning step involves transferring the chromosomes from said NT unit prior to activation to a second enucleated oocyte. DATED this 22 day of February 2002 UNIVERSITY OF MASSACHUSETTS WATERMARK PATENT TRADE MARK ATTORNEYS 290 BUR WOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA P18181AUOO CJH:BJD:SLB C. g.e 0 S e.g e.g. C g@ g e.g. 0B a *gg S. egg. C g CS S. go S CC S C 5* S. C 0 0 900* C. C S. S egg. a C 0* C SCC 0 S. C *4