WO2001019977A1 - Embryonic or stem-like cell lines produced by cross species nuclear transplantation and methods for enhancing embryonic development by genetic alteration of donor cells or by tissue culture conditions - Google Patents

Abstract

An improved method of nuclear transfer involving the transplantation of differentiated donor cell nuclei into enucleated oocytes of a species different from the donor cell is provided. The resultant nuclear transfer units are useful for the production of isogenic embryonic stem cells, in particular human isogenic embryonic or stem cells. These embryonic or stem-like cells are useful for producing desired differentiated cells and for introduction, removal or modification, of desired genes, e.g., at specific sites of the genome of such cells by homologous recombination. These cells, which may contain a heterologous gene, are especially useful in cell transplantation therapied and for in vitro study of cell differentiation. Also, methods for improving nuclear transfer efficiency by genetically altering donor cells to inhibit apoptosis, select for a specific cell cycle and/or enhance embryonic growth and development are provided.

Description

EMBRYONIC OR STEM-LIKE CELL LINES PRODUCED BY

CROSS SPECIES NUCLEAR TRANSPLANTATION AND

METHODS FOR ENHANCING EMBRYONIC DEVELOPMENT

BY GENETIC ALTERATION OF DONOR CELLS OR

BY TISSUE CULTURE CONDITIONS

CROSS-REFERENCE TO RELATES APPLICATIONS

This application claims priority under 35 U.S.C. §119 to PCT/US99/04608, filed

on March 2, 1999. Also, this application is a continuation-in-part of U.S. Serial No.

09/032,995, filed March 2, 1998, which is in turn a continuation-in-part of U.S. Serial No.

08/699,040, filed on August 19, 1996. All of these applications are incorporated by

reference in their entirety herein.

FIELD OF THE INVENTION

The present invention generally relates to the production of embryonic or stem-like

cells by the transplantation of cell nuclei derived from animal or human cells into

enucleated animal oocytes of a species different from the donor nuclei. The present

invention more specifically relates to the production of primate or human embryonic or

stem-like cells by transplantation of the nucleus of a primate or human cell into an

enucleated animal oocyte, e.g., a primate or ungulate oocyte and in a preferred

embodiment a bovine enucleated oocyte.

The present invention further relates to the use of the resultant embryonic or stem¬

like cells, preferably primate or human embryonic or stem-like cells for therapy, for diag-

nostic applications, for the production of differentiated cells which may also be used for therapy or diagnosis, and for the production of transgenic embryonic or transgenic

differentiated cells, cell lines, tissues and organs. Also, the embryonic or stem-like cells

obtained according to the present invention may themselves be used as nuclear donors in

nuclear transplantation or nuclear transfer methods for the production of chimeras or

clones, preferably transgenic cloned or chimeric animals.

BACKGROUND OF THE INVENTION

Methods for deriving embryonic stem (ES) cell lines in vitro from early

preimplantation mouse embryos are well known. (See, e.g., Evans et al., Nature, 29: 154-

156 (1981); Martin, Proc. Natl. Acad. Sci., USA, 78:7634-7638 (1981)). ES cells can be

passaged in an undifferentiated state, provided that a feeder layer of fibroblast cells

(Evans et al., Id.) or a differentiation inhibiting source (Smith et al., Dev. Biol, 121:1-9

(1987)) is present.

ES cells have been previously reported to possess numerous applications. For

example, it has been reported that ES cells can be used as an in vitro model for differen-

tiation, especially for the study of genes which are involved in the regulation of early

development. Mouse ES cells can give rise to germline chimeras when introduced into

preimplantation mouse embryos, thus demonstrating their pluripotency (Bradley et al.,

Nature, 309:255-256 (1984)).

In view of their ability to transfer their genome to the next generation, ES cells

have potential utility for germline manipulation of livestock animals by using ES cells

with or without a desired genetic modification. Moreover, in the case of livestock animals, e.g., ungulates, nuclei from like preimplantation livestock embryos support the

development of enucleated oocytes to term (Smith et al., Biol. Reprod., 40:1027-1035

(1989); and Keefer et al., Biol. Reprod., 50:935-939 (1994)). This is in contrast to nuclei

from mouse embryos which beyond the eight-cell stage after transfer reportedly do not

support the development of enucleated oocytes (Cheong et al, Biol. Reprod., 48:958

(1993)). Therefore, ES cells from livestock animals are highly desirable because they

may provide a potential source of totipotent donor nuclei, genetically manipulated or

otherwise, for nuclear transfer procedures.

Some research groups have reported the isolation of purportedly pluripotent

embryonic cell lines. For example, Notarianni et al., J. Reprod. Fert. Suppl, 43:255-260

(1991), report the establishment of purportedly stable, pluripotent cell lines from pig and

sheep blastocysts which exhibit some morphological and growth characteristics similar

to that of cells in primary cultures of inner cell masses isolated immunosurgically from

sheep blastocysts. (Id.) Also, Notarianni et al., J. Reprod. Fert. Suppl., 41:51-56 (1990)

discloses maintenance and differentiation in culture of putative pluripotential embryonic

cell lines from pig blastocysts. Further, Gerfen et al., Anim. Biotech, 6(1):1-14 (1995)

disclose the isolation of embryonic cell lines from porcine blastocysts. These cells are

stably maintained in mouse embryonic fibroblast feeder layers without the use of

conditioned medium. These cells reportedly differentiate into several different cell types

during culture (Gerfen et al., Id). Further, Saito et al., Roux's Arch. Dev. Biol, 201 : 134-141 (1992) report bovine

embryonic stem cell-like cell lines cultured which survived passages for three, but were

lost after the fourth passage. Still further, Handyside et al., Roux's Arch. Dev. Biol.,

196:185-190 (1987) disclose culturing of immunosurgically isolated inner cell masses of

sheep embryos under conditions which allow for the isolation of mouse ES cell lines

derived from mouse ICMs. Handyside et al. (1987) (Id.), report that under such condi¬

tions, the sheep ICMs attach, spread, and develop areas of both ES cell-like and

endoderm-like cells, but that after prolonged culture only endoderm-like cells are evident.

(Id.)

Recently, Cherny et al., Theriogenology, 41 :175 (1994) reported purportedly

pluripotent bovine primordial germ cell-derived cell lines maintained in long-term

culture. These cells, after approximately seven days in culture, produced ES-like colonies

which stain positive for alkaline phosphatase (AP), exhibited the ability to form embryoid

bodies, and spontaneously differentiated into at least two different cell types. These cells

also reportedly expressed mRNA for the transcription factors OCT4, OCT6 and HES1,

a pattern of homeobox genes which is believed to be expressed by ES cells exclusively.

Also recently, Campbell et al., Nature, 380:64-68 (1996) reported the production

of live lambs following nuclear transfer of cultured embryonic disc (ED) cells from day

nine ovine embryos cultured under conditions which 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 nuclear transfer and that totipotency is maintained in

culture.

Van Stekelenburg-Hamers et al., Mol. Reprod. Dev., 40:444-454 (1995), reported

the isolation and characterization of purportedly permanent cell lines 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 determine which feeder cells and culture

media are most efficient in supporting the attachment and outgrowth of bovine ICM cells.

They concluded based on their results that the attachment and outgrowth of cultured ICM

cells is enhanced 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¬

rum) to supplement the culture medium. Van Stekelenburg et al reported, however, that

their cell lines resembled epithelial cells more than pluripotent ICM cells. (Id.)

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 94/26889, published

November 24, 1994, report the isolation, selection and propagation of animal stem cells

which purportedly may be used to obtain transgenic animals. Also, Evans et al.,

WO 90/03432, published on April 5, 1990, reported the derivation of purportedly

pluripotent embryonic stem cells derived from porcine and bovine species which

assertedly are useful for the production of transgenic animals. Further, Wheeler et al,

WO 94/26884, published November 24, 1994, disclosed embryonic stem cells which are

assertedly useful for the manufacture of chimeric and transgenic ungulates. Thus, based on the foregoing, it is evident that many groups have attempted to produce ES cell lines,

e.g., because of their potential application in the production of cloned or transgenic

embryos and in nuclear transplantation.

The use of ungulate ICM cells for nuclear transplantation has also been reported.

For example, Collas et al., Mol. Reprod. Dev., 38:264-267 (1994) disclose nuclear trans¬

plantation of bovine ICMs by microi jection of the lysed donor cells into enucleated

mature oocytes. The reference disclosed culturing of embryos in vitro for seven days to

produce fifteen blastocysts which, upon transferral into bovine recipients, resulted in four

pregnancies and two births. Also, Keefer et al., Biol. Reprod., 50:935-939 (1994),

disclose the use of bovine ICM cells as donor nuclei in nuclear transfer procedures, to

produce blastocysts which, upon transplantation into bovine recipients, resulted in several

live offspring. Further, Sims et al., Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993),

disclosed the production of calves by transfer of nuclei from short-term in vitro cultured

bovine ICM cells into enucleated mature oocytes.

Also, the production of live lambs following nuclear transfer of cultured

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 transfer and the

production of chimeric fetuses has also been reported (Stice et al., Biol. Reprod., 54:100-

110 (1996)); Collas et al, Mol. Reprod. Dev., 38:264-267 (1994).

Further, there have been previous attempts to produce cross species NT units

(Wolfe et al., Theriogenology, 33:350 (1990). Specifically, bovine embryonic cells were fused with bison oocytes to produce some cross species NT units possibly having an inner

cell mass. However, embryonic cells, not adult cells were used, as donor nuclei in the

nuclear transfer procedure. The dogma has been that embryonic cells are more easily

reprogrammed than adult cells. This dates back to earlier NT studies in the frog (review

by DiBerardino, Differentiation, 17:17-30 (1980)). Also, this study involved very

phylogenetically similar animals (cattle nuclei and bison oocytes). By contrast, previ¬

ously when more diverse species were fused during NT (cattle nuclei into hamster

oocytes), no inner cell mass structures were obtained. Further, it has 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.

Also, Collas et al (Id.), taught the use of granulosa cells (adult somatic cells) to

produce bovine nuclear transfer embryos. However, unlike the present invention, these

experiments did not involve cross-species nuclear transfer. Also, unlike the present

invention ES-like cell colonies were not obtained.

Recently, U.S. Patent No. 5,843,780, issued to James A. Thomson on December

1, 1998, assigned to the Wisconsin Alumni Research Foundation, purports to disclose a

purified preparation of primate embryonic stem cells that are (i) capable of proliferation

in an in vitro culture for over one year; (ii) maintain a karyotype in which all

chromosomes characteristic of the primate species are present and not noticeably altered

through prolonged culture; (iii) maintains the potential to differentiate into derivatives of

endoderm, mesoderm and ectoderm tissues throughout culture; and (iv) will not differentiate when cultured on a fibroblast feeder layer. These cells were reportedly

negative for the 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

include the presence of all the chromosomes characteristic of the primate species and in

which none of the chromosomes are altered. Further, these cells are respectfully positive

for the TRA-1-60, and TRA-1-81 markers. The cells purportedly differentiate into

endoderm, mesoderm and ectoderm cells when injected into a SCID mouse. Also,

purported embryonic stem cell lines derived from human or primate blastocysts are

discussed in Thomson et al., Science 282:1145-1147 and Proc. Natl. Acad. Sci., USA

92:7844-7848 (1995).

Thus, Thomson disclose what purportedly are non-human primate and human

embryonic or stem-like cells and methods for their production. However, 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 their significant therapeutic and

diagnostic potential.

In this regard, numerous human diseases have been identified which may be

treated by cell transplantation. For example, Parkinson's disease is caused by degenera¬

tion of dopaminergic neurons in the substantia nigra. Standard treatment for Parkinson's

involves administration of L-DOPA, which temporarily ameliorates the loss of dopamine,

but causes severe side effects and ultimately does not reverse the progress of the disease.

A different approach to treating Parkinson's, which promises to have broad applicability to treatment of many 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 a variety of brain regions can be incorporated into the adult brain. Such

grafts have been shown to alleviate experimentally induced behavioral deficits, including

complex cognitive functions, in laboratory animals. Initial test results from human

clinical trials have also been promising. However, supplies of human fetal cells or tissue

obtained from miscarriages is very limited. Moreover, obtaining cells or tissues from

aborted fetuses is highly controversial.

There is currently no available procedure for producing "fetal-like" cells from the

patient. Further, allograft tissue is not readily available and both allograft and xenograft

tissue are subject to graft rejection. Moreover, in some cases, it would be beneficial to

make genetic modifications in cells or tissues before transplantation. However, many

cells or tissues wherein such modification would be desirable do not divide well in culture

and most types of genetic transformation require rapidly dividing cells.

There is therefore a clear need in the art for a supply of human embryonic or stem¬

like undifferentiated cells for use in transplants and cell and gene therapies.

OBJECTS OF THE INVENTION

It is an object of the invention to provide novel and improved methods for

producing embryonic or stem-like cells. 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 nucleus of

a mammalian or human cell into an enucleated oocyte of a different species.

It is another specific object of the invention to provide a novel method for

producing non-human primate or human embryonic or stem-like cells which involves

transplantation of the nucleus of a non-human primate or human cell into an enucleated

animal or human oocyte, e.g., an ungulate, human or primate enucleated oocyte.

It is another object of the invention to enhance the efficacy of cross-species nuclear

transfer by incorporating mitochondrial DNA derived from the same species (preferably

same donor) as the donor cell into the oocyte of a different species that is used for nuclear

transfer, before or after enucleation, or into the nuclear transfer unit (after the donor cell

has been introduced).

It is still another object of the invention to enhance the efficacy of cross-species

nuclear transfer by fusing an enucleated somatic cell (e.g., an enucleated human somatic

cell) (karyoplast) with an activated or non-activated, enucleated or non-enucleated oocyte

of a different species, e.g., bovine, or by fusion with an activated or unactivated cross-

species NT unit which may be cleaved or uncleaved.

It is another object of the invention to provide a novel method for producing

lineage-defective non-human primate or human embryonic or stem-like cells which

involves transplantation of the nucleus of a non-human primate or human cell, e.g., a

human adult cell into an enucleated non-human primate or human oocyte, wherein such cell has been genetically engineered to be incapable of differentiation into a specific cell

lineage or has been modified such that the cells are "mortal", and thereby do not give rise

to a viable offspring, e.g., by engineering expression of anti-sense or ribozyme telomerase

gene.

It is still another object of the invention to enhance efficiency of nuclear transfer

and specifically to enhance the development of preimplantation embryos produced by

nuclear transfer by genetically engineering donor somatic cells used for nuclear transfer

to provide for the expression of genes that enhance embryonic development, e.g., genes

of the MHC I family, and in particular Ped genes such as Q7 and/or Q9.

It is another object of the invention to enhance the production of nuclear transfer

embryos, e.g., cross-species nuclear transfer embryos, by the introduction of transgenes

before or after nuclear transfer that provide for the expression of an antisense DNA

encoding a cell death gene such as BAX, Apaf-1, or capsase, or a portion thereof, or

demethylase.

It is yet another object of the invention to enhance the production of nuclear

transfer embryos by TP and more specifically nuclear transfer embryos by genetically

altering the donor cell used for nuclear transfer such that it is resistant to apoptosis, e.g.,

by introduction of a DNA construct that provides for the expression of genes that inhibit

apoptosis, e.g., Bcl-2 or Bcl-2 family members and/or by the expression of antisense

ribozymes specific to genes that induce apoptosis during early embryonic development. It is still another object of the invention to improve the efficacy of nuclear transfer

by improved selection of donor cells of a specific cell cycle stage, e.g., Gl phase, by

genetically engineering donor cells such that they express a DNA construct encoding a

particular cyclin linked to a detectable marker, e.g., one that encodes a visualizable (e.g.,

fluorescent tag) marker protein.

It is also an object of the invention to enhance the development of in vitro

produced embryos, by culturing such embryos in the presence of one or more protease

inhibitors, preferably one or more capsase inhibitors, thereby inhibiting apoptosis.

It is another object of the invention to provide 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 a more specific object of the invention to provide 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, e.g., a human, primate or ungulate enucle-

ated oocyte.

It is another object of the invention to use such embryonic or stem-like cells for

therapy or diagnosis.

It is a specific object of the invention to use such primate or human embryonic or

stem-like cells for treatment or diagnosis of any disease wherein cell, tissue or organ

transplantation is therapeutically or diagnostically beneficial. It is another specific object of the invention to use the embryonic or stem-like cells

produced according to the invention for the production of differentiated cells, tissues or

organs.

It is a more specific object of the invention to use the primate or human embryonic

or stem-like cells produced accordmg to the invention for the production of differentiated

human cells, tissues or organs.

It is another specific object of the invention to use the embryonic or stem-like cells

produced according to the invention for the production of genetically engineered embry¬

onic or stem-like cells, which cells may be used to produce genetically engineered or

transgenic differentiated human cells, tissues or organs, e.g., having use in gene therapies.

It is another specific object of the invention to use the embryonic or stem-like cells

produced according to the invention in vitro, e.g. for study of cell differentiation and for

assay purposes, e.g. for drug studies.

It is another object of the invention to provide 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 replace-

ment, burns, vascular diseases, urinary tract diseases, as well as for the treatment of

immune defects, bone marrow transplantation, cancer, among other diseases. It is another object of the invention to use the transgenic or genetically engineered

embryonic or stem-like cells produced according to the invention for gene therapy, in

particular for the treatment and/or prevention of the diseases and injuries identified,

supra.

It is another object of the invention to use 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 as nuclear donors for nuclear

transplantation.

It is still another object of the invention to use genetically engineered ES cells

produced according to the invention for the production of transgenic animals, e.g., 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, e.g., therapeutics or nutripharmaceuticals.

With the foregoing and other objects, advantages and 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.

BRIEFS 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 (e.g., human) and oocytes of another non-related

species has never been reported. Rather, while embryos produced by fusion of cells of

closely related species, has been reported, e.g., 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

from aborted fetuses.

Also, prior to the present invention, no one obtained embryonic or stem-like cells

by cross-species nuclear transplantation. Quite unexpectedly, the present inventors discovered that human embryonic or

stem-like cells and cell colonies may be obtained by transplantation of the nucleus of a

human cell, e.g., an adult differentiated human cell, into an enucleated animal oocyte,

which is used to produce nuclear transfer (NT) units, the cells of which upon culturing

give rise to human embryonic or stem-like cells and cell colonies. This result is highly

surprising because it is the first demonstration of effective cross-species nuclear

transplantation involving the introduction of a differentiated donor cell or nucleus into an

enucleated oocyte of a genetically dissimilar species, e.g., the transplantation of cell

nuclei from a differentiated animal or human cell, e.g., adult cell, into the enucleated egg

of a different animal species, to produce nuclear transfer units containing cells which

when cultured under appropriate conditions give rise to embryonic or stem-like cells and

cell colonies.

Preferably, the NT units used to produce ES-like cells will be cultured to a size of

at least 2 to 400 cells, preferably 4 to 128 cells, and most preferably to a size of at least

about 50 cells.

In the present invention, embryonic or stem-like cells refer to cells 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 which they are typically

produced, i.e., by cross-species nuclear transfer. While these cells are expected to possess

similar differentiation capacity as normal stem cells they may possess some insignificant

differences because of the manner they 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 conventional embryonic stem cells.

The present discovery was made based on the observation that nuclear

transplantation of the nucleus of an adult human cell, specifically a human epithelial cell

obtained from the oral cavity of a human donor, when transferred 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 embryonic cells and human embryonic or stem¬

like cell colonies. This result has recently been reproduced by transplantation of

keratinocytes from an adult human into an enucleated bovine oocyte with the successful

production of a 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

must undergo maturation processes during embryonic development which are sufficiently

similar or conserved so as to permit the bovine oocyte to function as an effective sub¬

stitute or surrogate for a human oocyte. Apparently, oocytes in general comprise factors,

likely proteinaceous or nucleic acid in nature, that induce embryonic development under

appropriate conditions, and these functions that are the same or very similar in different

species. These factors may comprise material RNAs and/or telomerase.

Based on the fact that human cell nuclei can be effectively transplanted into bovine

oocytes, it is reasonable to expect that human cells may be transplanted into oocytes of

other non-related species, e.g., other ungulates as well as other animals. In particular,

other ungulate oocytes should be suitable, e.g. pigs, sheep, horses, goats, etc. Also, oocytes from other sources should be suitable, e.g. oocytes derived from other primates,

amphibians, rodents, rabbits, 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

blastocysts to produce human ES cells.

Therefore, in its broadest embodiment, the present invention involves the

transplantation of an animal or human cell nucleus or animal or human cell into an oocyte

(preferably enucleated) of an animal species different from the donor nuclei, by 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. Enucleation (removal of endogenous oocyte

nucleus) may be effected before or after nuclear transfer. For example, the invention may

involve the transplantation of an ungulate cell nucleus or ungulate cell into an enucleated

oocyte of another species, e.g., another ungulate or non-ungulate, by injection or fusion,

which cells and/or nuclei are combined to 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 preferably 4 to 128 cells, and most preferably at least about 50 cells.

The cells of such NT units may be used to produce embryonic or stem-like cells or cell

colonies upon culturing.

However, the preferred embodiment of the invention comprises the production of

non-human primate or human embryonic or stem-like cells by transplantation of the

nucleus of a donor human cell or a human cell into an enucleated human, primate, or non- primate animal oocyte, e.g., an ungulate oocyte, and in a preferred embodiment a bovine

enucleated oocyte.

In general, the embryonic or stem-like cells will be produced by a nuclear transfer

process comprising the following steps:

(i) obtaining desired human or animal cells to be used as a source of donor nuclei

(which may be genetically altered);

(ii) obtaining oocytes from a suitable source, e.g. a mammal and most preferably

a primate or an ungulate source, e.g. bovine,

(iii) enucleating said oocytes by removal of endogenous nucleus;

(iv) transferring the human or animal cell or nucleus into the enucleated oocyte

of an animal species different than the donor cell or nuclei, e.g., by fusion or injection,

wherein steps (iii) and (iv) may be effected in either order;

(v) culturing the resultant NT product or NT unit to produce multiple cell

structures (embryoid structures having a discernible inner cell mass); and

(vi) culturing cells obtained from said embryos to obtain embryonic or stem- like

cells and stem-like cell colonies.

Nuclear transfer techniques or nuclear transplantation techniques are known in the

literature and are described in many of the references cited in the 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, Biol. Reprod., 50:935-939 (1994);

Sims et al, Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993); WO 94/26884; WO 94/24274, and WO 90/03432, which are incorporated by reference in their entirety herein.

Also, U.S. Patent Nos. 4,944,384 and 5,057,420 describe procedures for bovine nuclear

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

Human or animal cells, preferably mammalian cells, may be obtained and cultured

by well known methods. Human and animal cells useful in the present invention include,

by way of example, epithelial, neural cells, epidermal cells, keratinocytes, hematopoietic

cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), other immune

cells, erythrocytes, macrophages, melanocytes, monocytes, mononuclear cells,

fibroblasts, cardiac muscle cells, and other muscle cells, etc. Moreover, the human cells

used for nuclear transfer may be obtained from different organs, e.g., skin, lung, pancreas,

liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and other

urinary organs, etc. These are just examples of suitable donor cells. Suitable donor cells,

i.e., cells useful in the subject invention, may be obtained from any cell or organ of the

body. This includes all somatic or germ cells. Preferably, the donor cells or nucleus

would comprise actively dividing, i.e., non-quiescent, cells as this has been reported to

enhance cloning efficacy. Also preferably, such donor cells will be in the Gl cell cycle.

The resultant blastocysts may be used to obtain embryonic stem cell lines

according to the culturing methods reported by Thomson et al., Science, 282:1145-1147

(1998) and Thomson et al., Proc. Natl. Acad. Sci., USA, 92:7544-7848 (1995),

incorporated by reference in their entirety herein. 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 human

keratinocytes. However, as discussed, the disclosed method is applicable to other human

cells or nuclei. Moreover, the cell nuclei may be obtained from both human somatic and

germ cells.

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 at various stages

have been thoroughly reviewed in U.S. Patent 5,262,409, which is herein incorporated

by reference. In particular, while cycloheximide has been reported to have an inhibitory

effect on mitosis (Bowen and Wilson (1955) J. Heredity, 45:3-9), it may also be

employed for improved activation of mature bovine follicular oocytes when combined

with electric pulse treatment (Yang et al. (1992) Biol. Reprod., 42 (Suppl. 1): 117).

Zygote gene activation is associated with hyperacetylation of Histone H4.

Trichostatin-A has been shown to inhibit histone deacetylase in a reversible manner

(Adenot et al. Differential H4 acetylation of paternal and maternal chromatin precedes

DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse

embryos. Development (Nov. 1997) 124(22): 4615-4625; Yoshida et al. Trichostatm A

and trapoxin: novel chemical probes for the role of histone acetylation in chromatin

structure and function. Bioessays (May, 1995) 17(5): 423-430), as have other compounds.

For instance, butyrate is also believed to cause hyper-acetylations of histones by

inhibiting histone deacetylase. Generally, butyrate appears to modify gene expression and in almost all cases its addition to cells in culture appears to arrest cell growth. Use of

butyrate in this regard is described in U.S. Patent No. 5,681,718, which is herein

incorporated by reference. Thus, donor cells may be 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 activation.

Additionally, demethylation of DNA is thought to be a requirement for proper

access of transcription factors to DNA regulatory sequences. Global demethylation of

DNA from the eight-cell stage to the blastocyst stage in preimplantation embryos has

previously been described (Stein et al., Mol. Reprod. & Dev., 47(4): 421-429). Also,

Jaenisch et al. (1997) have reported that 5-azacytidine can be used to reduce the level of

DNA methylation in cells, potentially leading to increased access of transcription factors

to DNA regulatory sequences. Accordingly, donor cells may be exposed to 5-azacytidine

(5-Aza) previous to fusion, or 5-Aza may be added to the culture medium from the 8 cell

stage to blastocyst. Alternatively, other known methods for effecting DNA demethylation

may be used.

The oocytes used for nuclear transfer may be obtained from animals including

mammals and amphibians. Suitable mammalian sources for oocytes include sheep,

bovines, ovines, pigs, horses, rabbits, goats, guinea pigs, mice, hamsters, rats, primates,

humans, etc. In the preferred embodiments, the oocytes will be obtained from primates

or ungulates, e.g., a bovine. Methods for isolation of oocytes are well known in the art. Essentially, this will

comprise isolating oocytes from the ovaries or reproductive tract of a mammal or

amphibian, e.g., a bovine. A readily available source of bovine oocytes is slaughterhouse

materials.

For the successful use of techniques such as genetic engineering, nuclear transfer

and cloning, oocytes must generally be matured in vitro before these cells may be used

as recipient cells for nuclear transfer, and before they can be fertilized by the sperm cell

to develop into an embryo. This process generally requires collecting immature

(prophase I) oocytes from animal ovaries, e.g., bovine ovaries obtained at a

slaughterhouse and maturing the oocytes in a maturation medium prior to fertilization or

enucleation until the oocyte attains the metaphase II stage, which in the case of bovine

oocytes generally occurs 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

calculation of time periods, "aspiration" refers to aspiration of the immature oocyte from

ovarian follicles .

Additionally, metaphase II stage oocytes, which have been matured in vivo have

been successfully used in nuclear transfer techniques. Essentially, mature metaphase II

oocytes are collected surgically from either non-superovulated or superovulated cows or

heifers 35 to 48 hours past the onset of estrus or past the injection of human chorionic

gonadotropin (hCG) or similar hormone. The stage of maturation of the oocyte at enucleation and nuclear transfer 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 mammalian embryo

cloning practices used metaphase II stage oocyte as the recipient oocyte because at this

stage it is believed that the oocyte can be or is sufficiently "activated" to treat the intro¬

duced nucleus as it does a fertilizing sperm. In domestic animals, and especially cattle,

the oocyte activation period generally ranges from about 16-52 hours, preferably about

28-42 hours post-aspiration.

For example, immature oocytes may be washed in HEPES buffered hamster

embryo culture medium (HECM) as described in Seshagine et al., Biol. Reprod. , 40, 544-

606, 1989, and then placed into drops of maturation medium consisting of 50 microliters

of tissue culture medium (TCM) 199 containing 10% fetal calf serum which contains

appropriate gonadotropins such as luteinizing hormone (LH) and follicle stimulating

hormone (FSH), and estradiol under a layer of lightweight paraffin or silicon at 39°C.

After a fixed time maturation period, which typically will range from about 10 to

40 hours, and preferably about 16-18 hours, the oocytes will typically be enucleated.

Prior to enucleation the oocytes will preferably be removed and placed in HECM

containing 1 milligram per milliliter of hyaluronidase prior to removal of cumulus cells.

This may be effected by repeated pipetting through very fine bore pipettes or by vortexing

briefly. The stripped oocytes are then 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 follows. As noted above, enucleation may be effected

before or after introduction of donor cell or nucleus because the donor nucleus is readily

discernible from endogenous nucleus.

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

No. 4,994,384 which is incorporated by reference herein. For example, metaphase II

oocytes are either placed in HECM, optionally containing 7.5 micrograms per milliliter

cytochalasin B, for immediate enucleation, or may be placed in a suitable medium, for

example CRlaa, plus 10% estrus cow serum, and then enucleated later, preferably not

more than 24 hours later, and more preferably 16-18 hours later.

Enucleation may be accomplished microsurgically using a micropipette 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 screening may be effected by

staining the oocytes with 1 microgram per milliliter 33342 Hoechst dye in HECM, and

then viewing the oocytes under ultraviolet irradiation for less than 10 seconds. The

oocytes that have been successfully enucleated can then be placed in a suitable culture

medium.

In the present invention, the recipient oocytes will typically be enucleated at a time

ranging from about 10 hours to about 40 hours after the initiation of in vitro maturation,

more preferably from about 16 hours to about 24 hours after initiation of in vitro matura-

tion, and most preferably about 16-18 hours after initiation of in vitro maturation. Enucleation may be effected before, simultaneous or after nuclear transfer. Also,

enucleation may be effected before, after or simultaneous to activation.

A single animal or human cell or nucleus derived therefrom which is typically

heterologous to the enucleated oocyte will then be transferred into the perivitelline space

of the oocyte, typically enucleated, used to produce the NT unit. However, removal of

endogenous nucleus may alternatively be effected after nuclear transfer. The animal or

human cell or nucleus and the enucleated oocyte will be used to produce NT units ac¬

cording to methods known in the art. For example, the cells may be fused by electro-

fusion. Electrofusion is accomplished by providing a pulse of electricity that is sufficient

to cause a transient break down of the plasma membrane. This breakdown of the plasma

membrane is very short because the membrane reforms rapidly. Essentially, if two

adjacent membranes are induced to break down, upon reformation the lipid bilayers

intermingle and small channels will open between the two cells. Due to the ther-

modynamic instability of such a small opening, it enlarges until the two cells become one.

Reference is made to U.S. Patent 4,997,384, by Prather et al., (incorporated by reference

in its entirety herein) for a further discussion of this process. A variety of electrofusion

media can be used including e.g., sucrose, mannitol, sorbitol and phosphate buffered

solution. Fusion can also be accomplished using Sendai virus as a fusogenic agent

(Graham, Wister lnot. Symp. Monogr., 9, 19, 1969).

Also, in some cases (e.g. with small donor nuclei) it may be preferable to inject the

nucleus directly into the oocyte rather than using electroporation fusion. Such techniques are disclosed in Collas and Barnes, Mol. Reprod. Dev., 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 μm

chamber by application of an electrical pulse of 90- 120V for about 15 μsec, about 24

hours after initiation of oocyte maturation. After fusion, the resultant fused NT units are

preferably placed in a suitable medium until activation, e.g., one identified infra. -

Typically activation will be effected shortly thereafter, typically less than 24 hours later,

and preferably about 4-9 hours later. However, it is also possible to activate the recipient

oocyte before or proximate (simultaneous) to nuclear transfer, which may or may not be

enucleated. For example, activation may be effected from about twelve hours prior to

nuclear transfer to about twenty-four hours after nuclear transfer. More typically,

activation is effected simultaneous or shortly after nuclear transfer, e.g., about four to nine

hours later.

The NT unit may be activated by known methods. Such methods include, e.g.,

culturing the NT unit at sub-physiological temperature, in essence by applying a cold, or

actually cool temperature shock to the NT unit. This may be most conveniently done by

culturing the NT unit at room temperature, which is cold relative to the physiological

temperature conditions to which embryos are normally exposed.

Alternatively, activation may be achieved by application of known activation

agents. For example, penetration of oocytes by sperm during fertilization has been shown

to activate prefusion oocytes to yield greater numbers of viable pregnancies and multiple genetically identical calves after nuclear transfer. Also, treatments such as electrical and

chemical shock or cycloheximide treatment may also be used to activate NT embryos

after fusion. Suitable oocyte activation methods are the subject of U.S. Patent No.

5,496,720, to Susko-Parrish et al., which is herein incorporated by reference.

5 For example, oocyte activation may be effected by simultaneously or sequentially:

(i) increasing levels of divalent cations in the oocyte, and

(ii) reducing phosphorylation of cellular proteins in the oocyte.

This will generally be effected by introducing divalent cations into the oocyte

cytoplasm, e.g., magnesium, strontium, barium or calcium, e.g., in the form of an iono-

0 phore. Other methods of increasing divalent cation levels include the use of electric

shock, treatment with ethanol and treatment with caged chelators.

Phosphorylation may be reduced by known methods, e.g., by the addition of kinase

inhibitors, e.g., serine-threonine kinase inhibitors, such as 6-dimethylamino-purine,

staurosporine, 2-arninopurine, and sphingosine.

5 Alternatively, phosphorylation of cellular proteins may be inhibited by

introduction of a phosphatase into the oocyte, e.g., phosphatase 2A and phosphatase 2B.

Specific examples of activation methods are listed below.

1. Activation by Ionomycin and DMAP

1- Place oocytes in Ionomycin (5 μM) with 2 mM of DMAP for 4

o minutes; 2- Move the oocytes into culture media with 2 mM of DMAP for 4

hours;

3- Rinse four times and place in culture.

2. Activation by Ionomycin DMAP and Roscovitin

1- Place oocytes in Ionomycin (5 μM) with 2 mM of DMAP for four

minutes;

2- Move the oocytes into culture media with 2 mM of DMAP and 200

microM of Roscovitin for three hours;

3- Rinse four times and place in culture.

3. Activation by exposure to Ionomycin followed by cytochalasin and

cycloheximide.

1- Place oocytes in Ionomycin (5 microM) for four minutes;

2- Move oocytes to culture media containing 5 μg/ml of cytochalasin

B and 5 μg/ml of cycloheximide for five hours;

3- Rinse four times and place in culture.

4. Activation by electrical pulses

1- Place eggs in mannitol media containing 100 μM CaCL₂;

2- Deliver three pulses of 1.0 kVcm^"1 for 20 μsec, each pulse 22

minutes apart;

3- Move oocytes to culture media containing 5 μg/ml of cytochalasin

B for three hours. 5. Activation by exposure with ethanol followed by cytochalasin and

cycloheximide

1- Place oocytes in 7% ethanol for one minute;

2- Move oocytes to culture media containing 5 μg/ml of cytochalasin

B and 5 μg/ml of cycloheximide for five hours;

3- Rinse four times and place in culture.

6. Activation by microinjection of adenophostine

1- Inject oocytes with 10 to 12 picoliters of a solution containing 10

μM of adenophostine;

2- Put oocytes in culture.

7. Activation by microinjection of sperm factor

1 - Inject oocytes with 10 to 12 picoliters of sperm factor isolated, e.g.,

from primates, pigs, bovine, sheep, goats, horses, mice, rats, rabbits

or hamsters;

2- Put eggs in culture.

8. Activation by microinjection of recombinant sperm factor.

9. Activation by Exposure to DMAP followed by Cycloheximide and Cytochalasin B

Place oocytes or NT units, typically about 22 to 28 hours post

maturation in about 2 mM DMAP for about one hour, followed by incubation for about two to twelve hours, preferably about eight hours, in

5 μg/ml of cytochalasin B and 20 μg/ml cycloheximide.

The above activation protocols are exemplary of protocols used for nuclear transfer

procedures, e.g., those including the use of primate or human donor cells or oocytes.

However, the above activation protocols may be used when either or both the donor cell

and nucleus is of ungulate origin, e.g., a sheep, buffalo, horse, goat, bovine, pig and/or

wherein the oocyte is of ungulate origin, e.g., sheet, pig, buffalo, horse, goat, bovine, etc.,

as well as for other species.

As noted, activation may be effected before, simultaneous, or after nuclear

transfer. In general, activation will be effected about 40 hours prior to nuclear transfer

and fusion to about 40 hours after nuclear transfer and fusion, more preferably about 24

hours before to about 24 hours after nuclear transfer and fusion, and most preferably from

about 4 to 9 hours before nuclear transfer and fusion to about 4 to 9 hours after nuclear

transfer and fusion. Activation is preferably effected after or proximate to in vitro or in

vivo maturation of the oocyte, e.g., approximately simultaneous or within about 40 hours

of maturation, more preferably within about 24 hours of maturation.

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 media suitable for

culturing and maturation of embryos are well known in the art. Examples of known

media, which may be used for bovine embryo culture and maintenance, include Ham's

F-10 + 10% fetal calf serum (FCS), Tissue Culture Medium- 199 (TCM-199) + 10% fetal calf serum, Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's Phosphate Buffered

Saline (PBS), Eagle's and Whitten's media. One of the most common media used for the

collection and maturation of oocytes is TCM-199, and 1 to 20% serum supplement

including fetal calf serum, newborn serum, estrual cow serum, lamb serum or steer

serum. A preferred maintenance medium includes TCM-199 with Earl salts, 10% fetal

calf serum, 0.2 Ma pyruvate and 50 μg/ml gentamicin sulphate. Any of the above may

also involve co-culture with a variety of cell types such as granulosa cells, oviduct cells,

BRL cells and uterine cells and STO cells.

In particular, human epithelial cells of the endometrium secrete leukemia

inhibitory factor (LIF) during the preimplantation and implantation period. 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 LIF for embryonic or stem¬

like cell cultures has been described in U.S. Patent 5,712,156, which is herein

incorporated by reference.

Another maintenance medium is described in U.S. Patent 5,096,822 to Rosenkrans,

Jr. et al., which is incorporated herein by reference. This embryo medium, named CR1,

contains the nutritional substances necessary to support an embryo. CR1 contains

hemicalcium L-lactate in amounts ranging from 1.0 mM to 10 mM, preferably 1.0 mM

to 5.0 mM. Hemicalcium L-lactate is L-lactate with a hemicalcium salt incorporated

thereon. Also, suitable culture medium for maintaining human embryonic cells in culture

as discussed in Thomson et al., Science, 282:1145-1147 (1998) and Proc. Nαtl. Acαd. Sci,

USA, 92:7844-7848 (1995).

Afterward, the cultured NT unit or units are preferably washed and then placed in

a suitable media, e.g., CRIaa medium, Ham's F-10, Tissue Culture Media -199 (TCM-

199). Tyrodes-Albumin-Lactate-Pyruvate (TALP) Dulbecco's Phosphate Buffered Saline

(PBS), Eagle's or Whitten's, preferably containing about 10% FCS. Such culturing will

preferably be effected in well plates which contain a suitable confluent feeder layer.

Suitable feeder layers include, by way of example, fibroblasts and epithelial cells, e.g.,

fibroblasts and uterine epithelial cells derived from ungulates, chicken fibroblasts, murine

(e.g., mouse or rat) fibroblasts, STO and SI-m220 feeder cell lines, and BRL cells.

In the preferred embodiment, the feeder cells will comprise mouse embryonic

fibroblasts. Means for preparation of a suitable fibroblast feeder layer are described in

the example which follows and is well within the skill of the ordinary artisan.

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-like cells or

cell colonies. Preferably, these NT units will be cultured until they reach a size of at least

about 2 to 400 cells, more preferably about 4 to 128 cells, and most preferably at least

about 50 cells. The culturing will be effected under suitable conditions, i.e., about 38.5 °C

and 5% C0₂, with the culture medium changed in order to optimize growth typically

about every 2-5 days, preferably about every 3 days. In the case of human cell/enucleated bovine oocyte derived NT units, sufficient

cells to produce an ES cell colony, typically on the order of about 50 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 donor, the species of the particular

oocyte, and culturing conditions. One skilled in the art can readily ascertain visually

when a desired sufficient number of cells has been obtained based on the morphology of

the cultured NT units.

In the case of human/human nuclear transfer embryos, or other embryos produced

using non-human primate donor or oocyte, it may be advantageous to use culture medium

known to be useful for maintaining human and other primate cells in tissue culture.

Examples of a culture media suitable for human embryo culture include the medium

reported in Jones et al, Human Reprod., 13(1):169-177 (1998), the Pl-catalog #99242

medium, and the P-l catalog #99292 medium, both available from Irvine Scientific, Santa

Ana, California, and those used by Thomson et al. (1998) and (1995), which references

are incorporated by reference in their entirety.

Another preferred medium comprises ACM + uridine + glucose + 1000 IU of LIF.

As discussed above, the cells used in the present invention will preferably

comprise mammalian somatic cells, most preferably cells derived from an actively

proliferating (non-quiescent) mammalian cell culture. In an especially preferred

embodiment, the donor cell will be genetically modified by the addition, deletion or

substitution of a desired DNA sequence. For example, the donor cell, e.g., a keratinocyte or fibroblast, e.g., of human, primate or bovine origin, may be transfected or transformed

with a DNA construct that provides for the expression of a desired gene product, e.g.,

therapeutic polypeptide. Examples thereof include lymphokines, e.g., IGF-I, IGF-II,

interferons, colony stimulating factors, connective tissue polypeptides such as collagens,

genetic factors, clotting factors, enzymes, enzyme inhibitors, etc.

Also, as discussed above, the donor cells may be modified prior to nuclear transfer,

e.g., to effect impaired cell lineage development, enhanced embryonic development

and/or inhibition of apoptosis. Examples of desirable modifications are discussed further

below.

One aspect of the invention will involve genetic modification of the donor cell,

e.g., a human cell, such that it is lineage deficient and therefore when used for nuclear

transfer it will be unable to give rise to a viable offspring. This is desirable especially in

the context of human nuclear transfer embryos, wherein 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 incapable of differentiating into specific cell

lineages when used for nuclear transfer. In particular, cells may be genetically modified

such that when used as nuclear transfer donors the resultant "embryos" do not contain or

substantially lack at least one of mesoderm, endoderm or ectoderm tissue.

This can be accomplished by, e.g., knocking out or impairing the expression of one

or more mesoderm, endoderm or ectoderm specific genes. Examples thereof include:

Mesoderm: SRF, MESP- 1 , HNF-4, beta-I integrin, MSD ; Endoderm: GATA-6, GATA-4;

Ectoderm: RNA helicase A, H beta 58.

The above list is intended to be exemplary and non-exhaustive of known genes

which are involved in the development of mesoderm, endoderm and ectoderm. The

generation of mesoderm deficient, endoderm deficient and ectoderm deficient cells and

embryos has been previously reported in the literature. See, e.g., Arsenian et al, EMBO

J., Vol. 17(2):6289-6299 (1998); Saga Y, Mech. Dev., Vol. 75(l-2):53-66 (1998);

Holdener et al, Development, Vol. 120(5): 1355-1346 (1994); Chen et al, Genes Dev. Vol.

8(20):2466-2477 (1994); Rohwedel et al, Dev. Biol., 201(2):167-189 (1998) (mesoderm);

Morrisey et al, Genes, Dev., Vol. 12(22):3579-3590 (1998); Soudais et al, Development,

Vol. 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. 111(3):801-811

(1991) (ectoderm).

In general, a desired somatic cell, e.g., a human keratinocyte, epithelial cell or

fibroblast, will be genetically engineered such that one or more genes 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., homologous

recombination. A preferred genetic system for effecting "knock-out" of desired genes is

disclosed by Capecchi et al, U.S. Patents 5,631,153 and 5,464,764, which reports

positive-negative selection (PNS) vectors that enable targeted modification of DNA

sequences in a desired mammalian genome. Such genetic modification will result in a cell that is incapable of differentiating into a particular cell lineage when used as a nuclear

transfer donor.

This genetically modified cell will be used to produce a lineage-defective nuclear

transfer embryo, i.e., that does not develop at least one of a functional mesoderm,

endoderm or ectoderm. Thereby, the resultant embryos, even if implanted, e.g., 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 in that they will produce cells of the one or

two remaining non-impaired lineage. For example, an ectoderm deficient human nuclear

transfer embryo will still give rise to mesoderm and endoderm derived differentiated

cells. An ectoderm deficient cell can be produced by deletion and/or impairment of one

or both of RNA helicase A or H beta 58 genes.

These lineage deficient donor cells may also be genetically modified to express

another desired DNA sequence.

Thus, the genetically modified donor cell will give rise to a lineage-deficient

blastocyst which, when plated, will differentiate into at most two of the embryonic germ

layers.

Alternatively, the donor cell can be modified such that it is "mortal". This can be

achieved by expressing anti-sense or ribozyme telomerase genes. This can be effected

by known genetic methods that will provide for expression of antisense DNA or

ribozymes, or by gene knockout. These "mortal" cells, when used for nuclear transfer,

will not be capable of differentiating into viable offspring. Another preferred embodiment of the present invention is the production of

nuclear transfer embryos that grow more efficiently in tissue culture. This is

advantageous in that it should reduce the requisite time and necessary fusions to 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 blastocysts and ES cells resulting

from nuclear transfer may have impaired development potential. While these problems

may often be alleviated by alteration of tissue culture conditions, an alternative solution

is to enhance embryonic development by enhancing expression of genes involved in

embryonic development.

For example, it has been reported that the gene products of the Ped type, which are

members of the MHC I family, are of significant importance to embryonic development.

More specifically, it has been reported in the case of mouse preimplantation embryos that

the Q7 and Q9 genes are responsible for the "fast growth" phenotype. Therefore, it is

anticipated that introduction of DNAs that provide for the expression of these and related

genes, or their human or other mammalian counterparts into donor cells, will give rise to

nuclear fransfer embryos that grow more quickly. This is particularly desirable in the

context of cross-species nuclear transfer embryos which may develop less efficiently in

tissue culture than nuclear transfer embryos produced by fusion of cells or nuclei of the

same species.

In particular, a DNA construct containing the Q7 and/or Q9 gene will be

introduced into donor somatic cells prior to nuclear transfer. For example, an expression construct can be constructed containing a strong constitutive mammalian promoter

operably linked to the Q7 and or Q9 genes, an IRES, one or more suitable selectable

markers, e.g,. neomycin, ADA, DHFR, and a poly-A sequence, e.g., bGH polyA

sequence. Also, it may be advantageous to further enhance Q7 and Q9 gene expression

by the inclusion of insulates. It is anticipated that these genes will be expressed early on

in blastocyst development as these genes are highly conserved in different species, e.g.,

bo vines, goats, porcine, dogs, cats, and humans. Also, it is anticipated that donor cells

can be engineered to affect other genes that enhance embryonic development. Thus, these

genetically modified donor cells should produce blastocysts and preimplantation stage

embryos more efficiently.

Still another aspect of the invention involves the construction of donor cells that

are resistant to apoptosis, i.e., programmed cell death. It has been reported in the

literature that cell death related genes are present in preimplantation stage embryos.

(Adams et al, Science, 281(5381): 1322-1326 (1998)). Genes reported to induce apoptosis

include, e.g., Bad, Bok, BH3, Bik, Hrk, BNIP3, Bim_L, Bad, Bid, and EGL-1. By contrast,

genes that reportedly protect cells from programmed cell death include, by way of

example, BcL-XL, Bcl-w, Mcl-1, Al, Nr-13, BHRF-1, LMW5-HL, ORF16, Ks-Bel-2,

E1B-19K, and CED-9.

Thus, donor cells can be constructed wherein genes that induce apoptosis are

"knocked out" or wherein the expression of genes that protect the cells from apoptosis is

enhanced or turned on during embryonic development. For example, this can be effected by introducing a DNA construct that provides

for regulated expression of such protective genes, e.g., Bcl-2 or related genes during

embryonic development. Thereby, the gene can be "turned on" by culturing the embryo

under specific growth conditions. Alternatively, it can be linked to a constitutive

promoter.

More specifically, a DNA construct containing a Bcl-2 gene operably linked to a

regulatable or constitutive promoter, e.g., PGK, SV40, CMV, ubiquitin, or beta-actin, an

IRES, a suitable selectable marker, and a poly-A sequence can be constructed and

introduced into a desired donor mammalian cell, e.g., human keratinocyte or fibroblast.

These donor cells, when used to produce nuclear transfer embryos, should be

resistant to apoptosis and thereby differentiate more efficiently in tissue culture. Thereby,

the speed and/or number of suitable preimplantation embryos produced by nuclear

transfer can be increased.

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 knock-out or by the use of

antisense or ribozymes against genes that are expressed in and which induce apoptosis

early on in embryonic development. Examples thereof are identified above. Cell death

genes that may be expressed in the antisense orientation include BAX, Apaf-1, and

capsases. Additionally, a transgene may be introduced that encodes for methylase or

demethylase in the sense or antisense orientation. DNAs that encode methylase and

demethylase enzymes are well known in the art. Still alternatively, donor cells may be constructed containing both modifications, i.e., impairment of apoptosis-inducing genes

and enhanced expression of genes that impede or prevent apoptosis. The construction and

selection of genes that affect apoptosis, and cell lines that express such genes, is disclosed

in U.S. Patent No. 5,646,008, which patent is incorporated by reference herein. Many

DNAs that promote or inhibit apoptosis have been reported and are the subject of

numerous patents.

Another means of enhancing cloning efficiency is to select cells of a particular cell

cycle stage as the donor cell. It has been reported that this can have significant effects on

nuclear transfer efficiency. (Barnes et al, Mol. Reprod. Devel., 36(1):33-41 (1993).

Different methods for selecting cells of a particular cell 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 chemical synchronization (Urbani et al, Exp. Cell Res.,

219(1):159-168 (1995). For example, a particular cyclin DNA may be operably linked

to a regulatory sequence, together with a detectable marker, e.g., green fluorescent protein

(GFP), followed by the cyclin destruction box, and optionally insulation 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 transfer donor. An example

thereof is the cyclin Dl gene in order to select for cells that are in Gl. However, any

cyclin gene should be suitable for use in the claimed invention. (See, e.g., King et al,

Mol. Biol. Cell, Vol. 7(9):1343-1357 (1996)). However, a less invasive or more efficient method for producing cells of a desired

cell cycle stage are needed. It is anticipated that this can be effected by genetically

modifying donor cells such that they express specific cyclins under detectable conditions.

Thereby, cells of a specific cell cycle can be readily discerned from other cell cycles.

Cyclins are proteins that are expressed only during specific stages of the cell cycle.

They include cyclin Dl, D2 and D3 in Gl phase, cyclin Bl and B2 in G2/M phase and

cyclin E, A and H in S phase. These proteins are easily translated and destroyed in the

cytogolcytosol. This "transient" expression of 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 to direct the prompt destruction of these proteins via the

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

In the present invention, donor cells will be constructed that express one or more

of such cyclin genes under easily detectable conditions, preferably visualizable, e.g., by

the use of a fluorescent label. For example, a particular cyclin DNA may be operably

linked to a regulatory sequence, together with a detectable marker, e.g., green fluorescent

protein (GFP), followed by the cyclin destruction box, and optionally insulation

sequences to enhance cyclin and/or marker protein expression. Thereby, cells of a

desired cell cycle can be easily visually detected and selected for use as a nuclear transfer

donor. An example thereof is the cyclin Dl gene which can be used to select for cells that

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

(See, e.g., King et al, Mol. Biol. Cell, Vol. 7(9):1343-1357 (1996)). As discussed, the present invention provides different methods for enhancing

nuclear transfer efficiency, preferably a cross-species nuclear transfer process. While the

present inventors have demonstrated that nuclei or cells of one species when inserted or

fused with an enucleated oocyte of a different species can give rise to nuclear transfer

embryos that produce blastocysts, which embryos can give rise to ES cell lines, the

efficiency of such process is quite low. Therefore, many 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. Yet another means for enhancing the development of nuclear transfer

embryos in vitro is by optimizing culture conditions. One means of achieving this result

will be to culture NT embryos under conditions impede apoptosis. 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 cell types. (See, Jurisicosva et al, Mol.

Reprod. Devel, 51(3):243-253 (1998).)

It is anticipated that blastocyst development will be enhanced by including in

culture media used for nuclear transfer and to maintain blastocysts or culture

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 inhibitor II,

capsase-9 inhibitor II, and capsase- 1 inhibitor I. The amount thereof will be an amount

effective to inhibit apoptosis, e.g., 0.00001 to 5.0% by weight of medium; more

preferably 0.01% to 1.0% by weight of medium. Thus, the foregoing methods may be used to increase the efficiency of nuclear transfer by enhancing subsequent blastocyst and

embryo development in tissue culture.

After NT units of the desired size are obtained, the cells are mechanically removed

from the zone and are then used to produce embryonic or stem-like cells and cell lines.

This is preferably effected by taking the clump of cells which comprise the NT unit,

which typically will contain at least about 50 cells, washing such cells, and plating the

cells onto a feeder layer, e.g., irradiated fibroblast cells. Typically, the cells used to

obtain the stem-like cells or cell colonies will be obtained from the inner most portion of

the cultured NT unit which is preferably at least 50 cells in size. However, NT units of

smaller or greater cell numbers as well as cells from other portions of the NT unit may

also be used to obtain ES-like cells and cell colonies.

It is further envisioned that a longer exposure of donor cell DNA to the oocyte 's

cytosol may facilitate the dedifferentiation process. This can be accomplished by re-

cloning, i.e., by taking blastomeres from a reconstructed embryo and fusing them with

a new enucleated oocyte. Alternatively, the donor cell may be fused with an enucleated

oocyte and four to six hours later, without activation, chromosomes removed and fused

with a younger oocyte. Activation would occur thereafter.

The cells are maintained in the feeder layer in a suitable growth medium, e.g.,

alpha MEM supplemented with 10% FCS and 0.1 mM beta-mercaptoethanol (Sigma) and

L-glutamine. The growth medium is changed as often as necessary to optimize growth,

e.g., about every 2-3 days. This culturing process results in the formation of embryonic or stem-like cells or

cell lines. In the case of human cell/bovine oocyte derived NT embryos, colonies are

observed by about the second day of culturing in the alpha MEM medium. However, this

time may vary dependent upon the particular nuclear 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 embryonic or stem-like cells. Other suitable media

are disclosed herein.

The embryonic or stem-like cells and cell colonies obtained will typically exhibit

an appearance similar to embryonic or stem-like cells of the species used as the nuclear

cell donor rather than the species of the donor oocyte. For example, in the case of embry¬

onic or stem-like cells obtained by the fransfer of a human nuclear donor cell into an

enucleated bovine oocyte, the cells exhibit a morphology more similar to mouse

embryonic stem cells than bovine ES-like cells.

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 appearance.

Further, the cell colony has a longer cell doubling time, about twice that of mouse ES

cells. Also, unlike bovine and porcine derived ES cells, the colony does not possess an

epithelial-like appearance.

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 (+), TRA-1-81 (+) and alkaline phosphatase (+). It is anticipated that human and primate ES cells produced

according to the present methods will exhibit similar or identical marker expression.

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 mesoderm, ectoderm

and endoderm tissues. This will be demonstrated by culturing ES cells produced

according to the invention under appropriate conditions, e.g., as disclosed by Thomsen,

U.S. Patent 5,843,780, incorporated by reference in its entirety herein. Alternatively, the

fact that the cells produced according to the invention are pluripotent will be confirmed

by injecting such cells into an animal, e.g., a SCID mouse, or large agricultural animal,

and thereafter obtaining tissues that result from said implanted cells. These implanted ES

cells should give rise to all different types of differentiated tissues, i.e., mesoderm,

ectoderm, and endodermal tissues.

The resultant embryonic or stem-like cells and cell lines, preferably human

embryonic or stem-like cells and cell lines, have numerous therapeutic and diagnostic

applications. Most especially, such embryonic or stem-like cells may be used for cell

transplantation therapies. Human embryonic or stem-like cells have application in the

treatment of numerous disease conditions.

Still another object of the present invention is to improve the efficacy of nuclear

transfer, e.g., cross-species nuclear transfer by introducing mitochondrial DNA of the

same species as the donor cell or nucleus into the recipient oocyte before or after nuclear

transfer, before or after activation, and before or after fusion and cleavage. Preferably, if the donor cell is human, human mitochondrial DNA will be derived from cells of the

particular donor, e.g., liver cells and tissue.

Methods for isolating mitochondria are well known in the art. Mitochondria can

be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will

depend upon the particular species of the donor cell. Examples of cells or tissues that

may be used as sources of mitochondria include fibroblasts, epithelium, liver, lung,

keratinocyte, stomach, heart, bladder, pancreas, esophageal, lymphocytes, monocytes,

mononuclear cells, cumulus cells, uterine cells, placental cells, intestinal cells,

hematopoietic cells, and tissues containing such cells.

For example, mitochondria can be isolated from tissue culture cells and rat liver.

It is anticipated that the same or similar procedures may be used to isolate mitochondria

from other cells and tissues. As noted above, preferred source of mitochondria comprises

human liver tissue because such cells contain a large number of mitochondria. Those

skilled in the art will be able to modify the procedure as necessary, dependent upon the

particular cell line or tissue. The isolated DNA can also be further purified, if desired,

known methods, e.g., density gradient centrifugation.

In this regard, it is known that mouse embryonic stem (ES) cells are capable of

differentiating into almost any cell type, e.g., hematopoietic stem cells. Therefore, human

embryonic or stem-like cells produced according to the invention should possess similar

differentiation capacity. The embryonic or stem-like cells according to the invention will

be induced to differentiate to 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 hematopoietic stem cells, muscle cells, cardiac muscle cells, liver cells,

cartilage cells, epithelial cells, urinary tract cells, etc., by culturing such cells in

differentiation medium and under conditions which provide for cell differentiation.

Medium and methods which result in the differentiation of embryonic stem cells are

known in the art as are suitable culturing conditions.

For example, Palacios et al, Proc. Natl. Acad. Sci, USA, 92:7530-7537 (1995)

teaches the production of hematopoietic stem cells from an embryonic cell line by

subjecting stem cells to an induction procedure comprising initially culturing aggregates

of such cells in a suspension culture medium lacking retinoic acid followed by culturing

in the same medium containing retinoic acid, followed by transferral of cell aggregates

to a substrate which provides for cell attachment.

Moreover, Pedersen, J. Reprod. Fertil. Dev., 6:543-552 (1994) is a review article

which references numerous articles disclosing methods for in vitro differentiation of

embryonic stem cells to produce various differentiated cell types including hematopoietic

cells, muscle, cardiac muscle, nerve cells, among others.

Further, Bain et al, Dev. Biol, 168:342-357 (1995) teaches in vitro differentiation

of embryonic stem cells to produce neural cells which possess neuronal properties. These

references are exemplary of reported methods for obtaining differentiated cells from

embryonic or stem-like cells. These references and in particular the disclosures therein relating to methods for differentiating embryonic stem cells are incorporated by reference

in their entirety herein.

Thus, using known methods and culture medium, one skilled in the art may culture

the subject embryonic or stem-like cells to obtain desired differentiated cell types, e.g.,

neural cells, muscle cells, hematopoietic cells, etc. In addition, the use of inducible Bcl-2

or Bcl-xl might be useful for enhancing in vitro development of specific cell lineages.

In vivo, Bcl-2 prevents 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 used to inhibit apoptosis of relevant cell lineages following transfection of donor cells

is disclosed in U.S. Patent No. 5,646,008, which is herein incorporated by reference.

The subject embryonic or stem-like cells may be used to obtain any desired

differentiated cell type. Therapeutic usages of such differentiated human cells are

unparalleled. For example, human hematopoietic stem cells may be used in medical

treatments requiring bone marrow transplantation. Such procedures are used to treat

many diseases, e.g., late stage cancers such as ovarian cancer and leukemia, as well as

diseases that compromise the immune system, such as AIDS. Hematopoietic stem cells

can be obtained, e.g., by fusing adult somatic cells of a cancer or AIDS patient, e.g.,

epithelial cells or lymphocytes with an enucleated oocyte, e.g., bovine oocyte, obtaining

embryonic or stem-like cells as described above, and culturing such cells under conditions which favor differentiation, until hematopoietic stem cells are obtained. Such

hematopoietic cells may be used in the treatment of diseases including cancer and AIDS.

Alternatively, adult somatic cells from a patient with a neurological disorder may

be fused with an enucleated animal oocyte, e.g., a primate or bovine oocyte, human

embryonic or stem-like cells obtained therefrom, and such cells cultured under

differentiation conditions to produce neural cell lines. Specific diseases treatable by

transplantation of such human neural cells include, by way of example, Parkinson's

disease, Alzheimer's disease, ALS and cerebral palsy, among others. In the specific case

of Parkinson's disease, it has been demonstrated that transplanted fetal brain neural cells

make the proper connections with surrounding cells and produce dopamine. This can

result in long-term reversal of Parkinson's disease symptoms.

To allow for specific selection of differentiated cells, donor cells may be

transfected with selectable markers expressed via inducible promoters, thereby permitting

selection or enrichment of particular cell lineages when differentiation is induced. For

example, CD34-neo may be used for selection of hematopoietic cells, Pwl-neo for

muscle cells, Mash-1-neo for sympathetic neurons, Mal-neo for human CNS neurons of

the grey matter of the cerebral cortex, etc.

The great advantage of the subject invention is that it provides an essentially

limitless supply of isogenic or synegenic human cells suitable for transplantation.

Therefore, it will obviate the significant problem associated with current transplantation

methods, i.e., rejection of the transplanted tissue which may occur because of host-vs- graft or graft-vs-host rejection. Conventionally, rejection is prevented or reduced by the

administration of anti-rejection drugs such as cyclosporin. However, such drugs have

significant adverse side-effects, e.g., immunosuppression, carcinogenic properties, as well

as being very expensive. The present invention should eliminate, or at least greatly

reduce, the need for anti-rejection drugs, such as cyclosporine, imulan, FK-506,

glucocorticoids, and rapamycin, and derivatives thereof.

Other diseases and conditions treatable by isogenic cell therapy include, by way

of example, spinal cord injuries, multiple sclerosis, muscular dystrophy, diabetes, liver

diseases, i.e., hypercholesterolemia, heart diseases, cartilage replacement, burns, foot

ulcers, gastrointestinal diseases, vascular diseases, kidney disease, urinary tract disease,

and aging related diseases and conditions.

Also, human embryonic or stem-like cells produced according to the invention

may be used to produce genetically engineered or transgenic human 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 endogenous gene or genes of human

embryonic or stem-like cells produced according to the invention, and allowing such cells

to differentiate into the desired cell type. A preferred method for achieving such

modification is by homologous recombination because such technique can be used to

insert, delete or modify a gene or genes at a specific site or sites in the stem-like cell

genome. This methodology can be used to replace defective genes, e.g., defective immune

system genes, cystic fibrosis genes, or to infroduce genes which result in the expression

of therapeutically beneficial proteins such as growth factors, lymphokines, cytokines,

enzymes, etc. For example, the gene encoding brain derived growth factor may be

infroduced into human embryonic or stem-like cells, the cells differentiated into neural

cells and the cells transplanted into a Parkinson's patient to retard the loss of neural cells

during such disease.

Previously, cell types transfected with BDNF varied from primary cells to

immortalized cell lines, either neural or non-neural (myoblast and fibroblast) derived

cells. For example, asfrocytes have been transfected with BDNF gene using retro viral

vectors, and the cells grafted into a rat model of Parkinson's disease (Yoshimoto et al.,

Brain Research, 691:25-36, (1995)).

This ex vivo therapy reduced Parkinson's-like symptoms in the rats up to 45% 32

days after transfer. Also, the ryrosine hydroxylase gene has been placed into astrocytes

with similar results (Lundberg et al., Develop. Neurol, 139:39-53 (1996) and references

cited therein).

However, such ex vivo systems have problems. In particular, refroviral vectors

currently used are down-regulated in vivo and the fransgene is only transiently expressed

(review by Mulligan, Science, 260:926-932 (1993)). Also, such studies used primary

cells, asfrocytes, which have finite life span and replicate slowly. Such properties

adversely affect the rate of transfection and impede selection of stably transfected cells. Moreover, it is almost impossible to propagate a large population of gene targeted

primary cells to be used in homologous recombination techniques.

By contrast, the difficulties associated with retroviral systems should be eliminated

by the use of human embryonic or stem-like cells. It has been demonstrated previously

by the subject assignee that cattle and pig embryonic cell lines can be transfected and

selected for stable integration of heterologous DNA. Such methods are described in

commonly assigned U.S. Serial No. 08/626,054, filed April 1, 1996, now U.S. Patent No.

5,905,042, incorporated by reference in its entirety. Therefore, using such 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 cell types, e.g., hematopoietic

cells, neural cells, pancreatic cells, cartilage cells, etc.

Genes which may be introduced into the subject embryonic or stem-like cells

include, by way of example, epidermal growth factor, basic fibroblast growth factor, glial

derived neurotrophic growth factor, insulin-like growth factor (I and II), neurotrophin-3,

neurotrophin-4/5, ciliary neurotrophic factor, AFT-1, cytokine genes (interleukins,

interferons, colony stimulating factors, tumor necrosis factors (alpha and beta), etc.),

genes encoding therapeutic enzymes, collagen, human serum albumin, etc.

In addition, it is also possible to use one of the negative selection systems now

known in the art for eliminating therapeutic cells from a patient if necessary. For

example, donor cells fransfected with the thymidine kinase (TK) gene will lead to the

production of embryonic cells containing the TK gene. 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 eliminated at any time from a patient upon gancyclovir

administration. Such a negative selection system is described in U.S. Patent No.

5,698,446, and is herein incorporated by reference.

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 genes which are

involved in the regulation of early development.

Also, differentiated cell tissues and organs using the subject embryonic or stem¬

like cells may be used in drug studies.

Further, the subject cells may be used to express recombinant DNAs.

Still further, the subject embryonic or stem-like cells may be used as nuclear

donors for the production of other embryonic or stem-like cells and cell colonies.

Also, cultured inner cell mass, or stem cells, produced according to the invention

may be introduced into animals, e.g., SCID mice, cows, pigs, e.g., under the renal capsule

or intramuscularly and used to produce a teratoma therein. This teratoma can be used to

derive different tissue types. Also, the inner cell mass produced by X-species nuclear

transfer may be introduced together with a biodegradable, biocompatible polymer matrix

that provides for the formation of 3-dimensional tissues. After tissue formation, the

polymer degrades, ideally just leaving the donor tissue, e.g., cardiac, pancreatic, neural,

lung, liver. In some instances, it may be advantageous to include growth factors and

proteins that promote angiogenesis. Alternatively, the formation of tissues can be effected totally in vitro, with appropriate culture media and conditions, growth factors,

and biodegradable polymer matrices.

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

provided.

EXAMPLE 1

MATERIALS AND METHODS

Donor Cells for Nuclear Transfer

Epithelial cells were lightly scraped from the inside of the mouth of a consenting

adult with a standard glass slide. The cells were washed off the slide into a petri dish

containing phosphate buffered saline without Ca or Mg. The cells were pipetted through

a small-bore pipette to break up cell clumps into a 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 transfer into enucleated cattle oocytes.

Nuclear Transfer Procedures

Basic nuclear transfer procedures have been described previously. Briefly, after

slaughterhouse oocytes were matured in vitro the oocytes were stripped of cumulus cells

and enucleated with a beveled micropipette at approximately 18 hours post maturation

(hpm). Enucleation was confirmed in TL-HEPES medium plus bisbenzimide (Hoechst

33342, 3 μg/ml; Sigma). Individual donor cells were then placed into the perivitelline

space of the recipient oocyte. 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 μsec was applied to the NT unit. This occurred at 24 hours post-initiation of

maturation (hpm) of the oocytes. The NT units were placed in CRlaa medium until 28

hpm.

The procedure used to artificially activate oocytes has been described 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 ionomycin (5 μM; CalBiochem, La Jolla,

CA) in TL-HEPES supplemented with 1 mg/ml BSA and then washed for five rnin in TL-

HEPES supplemented with 30 mg/ml BSA. The NT units were then transferred into a

microdrop of CRlaa culture medium containing 0.2 mM DMAP (Sigma) and cultured at

38.5 °C 5% C0₂ for four to five hours. The NT units were washed and then placed in a

CRlaa medium plus 10% FCS and 6 mg/ml BSA in four well plates containing a

confluent feeder layer of mouse embryonic fibroblasts (described below). The NT units

were cultured for three more days at 38.5° C and 5% C0₂. The culture medium was

changed every three days until day 12 after the time of activation. At this time NT units

reaching the desired cell number, i.e., about 50 cell number, were mechanically removed

from the zona and used to produce embryonic cell lines. A photograph of an NT unit

obtained as described above is contained in Figure 1.

Fibroblast feeder layer

Primary cultures of embryonic fibroblasts were obtained from 14-16 day old

murine fetuses. After the head, liver, heart and alimentary tract were aseptically removed,

the embryos were minced and incubated for 30 minutes at 37 °C in pre- warmed trypsin EDTA solution (0.05% trypsin/0.02% EDTA; GIBCO, Grand Island, NY). Fibroblast

cells were plated in tissue culture flasks and cultured in alpha-MEM medium

(BioWhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (FCS)

(Hyclone, Logen, UT), penicillin (100 IU/ml) and streptomycin (50 μl/ml). Three to four

days after passage, embryonic fibroblasts, in 35 x 10 Nunc culture dishes (Baxter

Scientific, McGaw Park, EL), were irradiated. The irradiated fibroblasts were grown and

maintained in a humidified atmosphere with 5% C0₂ in air at 37°C. The culture plates

which had a uniform monolayer of cells were then used to culture embryonic cell lines.

Production of embryonic cell line.

NT unit cells obtained as described above were washed and plated directly onto

irradiated feeder fibroblast cells. These cells included those of the inner 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-mercaptoethanol (Sigma). Growth

medium was exchanged every two to three days. The initial colony was observed by the

second day of culture. The colony was propagated and exhibits a similar morphology to

previously disclosed mouse embryonic stem (ES) cells. Individual cells within the colony

are not well defined and the perimeter of the colony is refractile and smooth in ap¬

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

cells. Also, unlike bovine and porcine derived ES cells, the colony does not have an

epithelial appearance thus far. Figures 2 through 5 are photographs of ES-like cell

colonies obtained as described, supra. Production of Differentiated Human Cells

The human embryonic cells obtained are transferred to a differentiation medium

and cultured until differentiated human cell types are obtained.

RESULTS

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

TABLE 1

The one NT unit that developed a structure having greater than 16 cells was plated

down onto a fibroblast feeder layer. This structure was attached to the feeder layer and

started to propagate forming a colony with a ES cell-like morphology (See, e.g., Figure

2). Moreover, although the 4 to 16 cell stage structures were not used to try and produce

an ES cell colony, it has been previously 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, Stice et al., 1996)). Therefore, it is expected that 4 - 16 cell stage NT

units should also give rise to embryonic or stem-like cells and cell colonies. Also, similar results were obtained upon fusion of an adult human keratinocyte cell

line with an enucleated bovine oocyte, which was cultured in media comprising ACM,

uridine, glucose, and 1000 IU of LLF. Out of 50 reconstructed embryos, 22 cleaved and

one developed into a blastocyst at about day 12. This blastocyst was plated and the

production of an ES cell line is ongoing.

EXAMPLE 2

A. Mitochondria Isolation Protocol from a Cell

This Example relates to isolation of mitochondria and use thereof to enhance the

efficiency of cross-species nuclear transfer. The number of mitochondria per cell varies

from cell line to cell line. For example, mouse L cells contain - 100 mitochondria per

cell, whereas there are at least twice that number in HeLa cells. The cells are swollen in

a hypotonic buffer and ruptured with a few strokes in a Dounce homogenizer using a

tight-fitting pestle, and the mitochondria are isolated by differential centrifugation.

The solutions, tubes, and homogenizer should be pre-chilled on ice. All

centrifugation steps are at 40 °C. This protocol is based on starting with a washed cell

pellet of 1-2 ml. The cell pellet is resuspended in 11 ml of ice-cold RSB and transferred

to a 16 ml Dounce homogenizer.

RSB Buffer

RSB (A hypotonic buffer for swelling the tissue culture cells)

lO mM NaCl

1.5 mM MgCl₂ 10 mM Tris-HCl, pH 7.5

MgCl₂

The cells are allowed to swell for five to ten minutes. The progress of the swelling

is maintained using a phase contrast microscope. The swollen cells are replaced,

preferably by several strokes with a pestle. Immediately after, 8 ml of 2.5x MS buffer are

added to give a final concentration of lx MS. The top of the homogenizer is then covered

with Parafilm and mixed by inverting a couple of times.

2.5x MS Buffer

525 mM mannitol

175 Mm sucrose

215 mM EDTA pH 7.5

lx MS Buffer

210 mM mannitol

70 mM Sucrose

5 mM Tris-HCl, pH 7.5

1 mM EDTA, pH 7.5

MS Buffer is an iso-osmotic buffer to maintain the tonicity of the

organelles and prevent agglutination.

Thereafter, the homogenate is transferred to a centrifuge tube for differential

centrifugation. The homogenizer is rinsed with a small amount of MS buffer and added to the homogenate. The volume is brought to 30 ml with MS buffer. The homogenate

is then centrifuged at 1300 g for five minutes to remove nuclei, unbroken cells, and large

membrane fragments. The supernatant is then poured into a clean centrifuge tube. The

nuclear spin-down is repeated twice. The supernatant is then transferred to a clean

centrifuge tube and a pellet containing the mitochondria is centrifuged at 17,000 g for 15

minutes. The supernatant is discarded and the inside of the tube wiped with a Kimwipe.

The mitochondria is washed by re-suspending the pellet in IX MS and repeating the

17,000 g sedimentation. The supernatant is discarded and the pellet is resuspended in a

buffer. Mitochondria can be stored at -80 °C for prolonged periods, e.g., up to a year, but

preferably will be used shortly thereafter for NT.

This basic protocol can be modified. In particular, it may be desirable to further

isolate mitochondrial DNA and us same for NT. In such case, contamination with nuclei,

not small organelles, potentially is a problem and the following modifications may be

made. For example, the cells may be harvested in stationary growth phase when the

fewest cells are actively dividing, and CaCl₂ substituted for MgCl₂ in the RSB to stabilize

the nuclear membrane. The washing of the mitochondrial pellet is omitted as is the

density gradient purification. Instead, the mitochondrial pellet is simply resuspended and

lysed, and the mitochondrial DNA purified from any remaining nuclear DNA. As noted,

suitable methods for purifying mitachondria and mitochondrial DNA are well known in

the art. Homogenization works best if the cells are resuspended in at least 5-1 OX the

volume of the cell pellet and if the cell suspension fills the homogenizer at least half full.

Press the homogenizer pestle straight down the tube, maintaining a firm, steady pressure.

The Dounce homogenizer disrupts swollen tissue culture cells by pressure change. As

the pestle is pressed down, pressure around the cell increases; when the cell slips past the

end of the pestle, the sudden decrease in pressure causes the cell to rupture. If the pestle

is very tight fitting, there may be some mechanical breakage as well.

B. Isolation of Mitochondria From Tissue

A mitochondrial isolation protocol is selected based on the particular tissue. For

example, the homogenization buffer should be optimized for the tissue, and the optimal

way to homogenize the tissue utilized. Suitable methods are well known in the art.

Rat liver is the most frequently used tissue for mitochondrial preparations because

it is readily available, is easy to homogenize, and the cells contain a large number of

mitochondria ( 1000-2000 per cell). For example, a motor-driven, Teflon and glass Potter-

Elvehjem homogenizer can be used homogenize rat liver. Alternatively, if the tissue is

soft enough, a Dounce homogenizer with a loose pestle can be used. The yield and purity

of the mitochondrial preparation is influenced by the method of preparation, speed of

preparation, and the age and physiological condition of the animal. As noted, methods

of purifying mitochondria are well known. Preferably, the buffer, tubes, and homogenizer will be pre-chilled. Pre-chilling a

glass and Teflon type homogenizer creates the proper gap between the tube and pestle.

The centrifugation steps are preferably effected at 40 °C.

Essentially, the process will comprise removal of the liver, taking care not to

rupture the gall bladder. This is placed in a beaker on ice and any connective tissue is

removed. The tissue is recognized and returned to the beaker, e.g., using very sharp

scissors, a scalpel, or razor blade, mince it into 1-2 slices. The pieces are then rinsed,

preferably twice, with homogenization buffer (IX MS) to remove most of the blood, and

the tissue transferred to the homogenizer tube. Enough homogenization buffer if added

to prepare a 1 : 10 (w/v) homogenate.

Use of Isolated Mitochondria or Mitochondrial DNA to Enhance NT Efficacy

It is theorized by the inventors that the efficacy of cross-species nuclear transfer

may be enhanced by introduction of mitochondria or mitochondrial DNA at the same

species as donor cell or nucleus. Thereupon, the nucleus DNA of resultant NT units will

be species compatible.

Mitochondria isolated by the above or other known procedures are incorporated,

typically by injection, into any of the following (in the case of human donor cell/bovine

oocyte nuclear transfer):

(i) non-activated, non-enucleated bovine oocytes;

(ii) non-activated, enucleated bovine oocytes;

(iii) activated, enucleated bovine oocytes; (iv) non-activated, fused (with human donor cell or nucleus"! bovine nnr.vtes-

(v) activated, fused and cleaved reconstructed (cow oocyte/human cell)

embryo; or

(vi) activated, fused one cell reconstructed (cow oocyte/human cell) embryo.

The same procedures will enhance other cross-species NTs. Essentially,

mitochondria will again be introduced into any of (i)-(vi) of the same species as the donor

cell or nucleus, and the oocyte will be of a different species origin. Generally about 1 to

200 picoliters of mitochondrial suspension are injected into any of the above. The

introduction of such mitochondria will result in NT units wherein the mitochondrial and

donor DNA are compatible.

EXAMPLE 3

Another method for improving the efficacy of the cross-species nuclear transfer

comprises the fusion of one or more enucleated somatic cells, typically human (of the

same species as donor cell or nucleus), with any of the following:

(i) non-activated, non-enucleated (e.g., bovine) oocyte;

(ii) non-activated, enucleated (e.g., bovine) oocyte;

(iii) activated, enucleated (e.g., bovine) oocyte;

(iv) non-activated, fused (with human cell) oocyte (typically bovine);

(v) activated, fused and cleaved reconstructed (e.g., cow oocyte/human cell)

embryo; (vi) activated, fused one cell reconstructed (cow oocyte/human cell) embryo;

or

(vii) non-activated, fused (e.g., with human cell) oocyte (typically bovine

oocyte).

Fusion is preferably effected by electrical pulse or by use of Sendai virus.

Methods for producing enucleated cells (e.g., human cells) are known in the art. A

preferred protocol is set forth below.

Enucleation Procedures:

Methods for the large-scale enucleation of cells with cytochalasin B are well

known in the art. Enucleation is preferably effected using the monolayer technique. This

method uses small numbers of cells attached to the growth surface of a culture disc and

is ideal if limited numbers of donor cells are available. Another suitable procedure, the

gradient technique, requires centrifugation of cells through Ficoll gradients and is best

suited for enucleation of large number (>10⁷) of cells.

Monolayer Technique. The monolayer technique is ideal for virtually any cell

which grows attached to the growth surface.

Polycarbonate or polypropylene 250-ml wide-mouth centrifuge bottles with screw-

top caps are sterilized by autoclaving. The caps preferably are autoclaved separately from

the bottle to prevent damage to the centrifuge bottle. The bottle are prepared for the

enucleation procedure by the sterile addition of 30 ml DMEM, 2 ml bovine serum, and 0.32 ml cytochalasin B (1 mg/ml) to each. The caps are placed on the bottles, and the

bottles are maintained at 37° prior to use.

The cells to be enucleated (from a few hundred to ~ 10⁵ cells) are seeded on a

culture dish (35 x 15 mm; Nunc Inc., Naperville, IL). Typically, the cells are grown for

at least twenty-four hours on the dishes to promote maximal attachment to the growth

surface. Preferably, the cells are prevented from becoming confluent. The culture dish

is prepared for centrifugation by wiping the outside of the bottom half of the dish

(containing the cells) with 70% (v/v) ethanol for the purpose of sterilization.

Alternatively, the dish can be kept sterile during cell culturing by maintaining it within

a larger, sterile culture dish. The medium is removed from the dish and the dish (without

top) is placed upside down within the centrifuge bottle.

The rotor (GSA, DuPont, Wilmington, DE) and centrifuge are preferably pre-

warmed to 37° by centrifugation for 30-45 minutes at 8000 rpm. The HS-4 swinging-

bucket rotor (DuPont) can alternatively be used. The optimal time and speed of

centrifugation varies for each cell type. For myoblasts and fibroblasts, the centrifuge

bottle with the culture dish is placed in the pre-warmed rotor and centrifuged for

approximately 20 minutes (interval between the time when the rotor reaches the desired

speed and the time when the centrifuge is turned off). Preferably, speeds of 6500 to 7200

rpm are used.

After centrifugation, the centrifuge bottle is removed from the rotor, and the

culture plate is removed from the bottles with forceps. A small amount of medium is maintained in the plate to keep the cells moist in order to maintain cell viability. The

outside of the dish, including the top edge, is wiped with a sterile wiper, then moistened

with 95% (v/v) ethanol, to remove any medium and to dry it. A sterile top is placed onto

the dish. If the enucleated cells are not going to be used immediately, complete culture

medium (medium supplemented with the appropriate concentration of serum) should be

added to the dish, and the cells placed in a C0₂ incubator. The resultant enucleated cells

(karyoplast) are fused with any of (i) - (viii) above.

While the present invention has been described and illustrated herein by reference

to various specific materials, procedures, and examples, it is understood that the invention

is not restricted to the particular material, combinations 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.

Claims

WHAT IS CLAIMED IS:

1. A method of producing embryonic or stem-like cells comprising the

following steps:

(i) 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 nuclear transfer unit;

(iii) culturing said activated nuclear transfer unit until greater than the 2-

cell developmental stage; and

(iv) culturing cells obtained from said cultured NT units to obtain

embryonic or stem-like cells.

2. The method of Claim 1 , wherein the cell inserted into the enucleated animal

oocyte is a human cell.

3. The method of Claim 2, wherein said human cell is an adult cell.

4. The method of Claim 2, wherein said human cell is an epithelial cell,

keratinocyte, lymphocyte or fibroblast.

5. The method of Claim 2, wherein the oocytes are obtained from a mammal.

6. The method of Claim 5, wherein the animal oocyte is obtained from an

ungulate.

7. The method of Claim 6, wherein said ungulate is selected from the group

consisting of bovine, ovine, porcine, equine, caprine, and buffalo.

8. The method of Claim 1, wherein the enucleated oocyte is matured prior to

enucleation.

9. The method of Claim 1, wherein the fused nuclear transfer units are

activated in vitro.

10. The method of Claim 1, wherein the activated nuclear transfer units are

cultured on a feeder layer culture.

11. The method of Claim 10, wherein the feeder layer comprises fibroblasts.

12. The method of Claim 1 , wherein in step (i v) cells from a NT unit having 16

cells or more are cultured on a feeder cell layer.

13. The method of Claim 12, wherein said feeder cell layer comprises

fibroblasts.

14. The method of Claim 13, wherein said fibroblasts comprise mouse

embryonic fibroblasts.

15. The method of Claim 1 , wherein the resultant embryonic or stem-like cells

are induced to differentiate.

16. The method of Claim 2, wherein the resultant embryonic or stem-like cells

are induced to differentiate.

17. The method of Claim 1 , wherein fusion is effected by electrofusion.

18. Embryonic or stem-like cells obtained according to the method of Claim 1.

19. Human embryonic or stem-like cells obtained according to the method of

Claim 2.

20. Human embryonic or stem-like cells obtained according to the method of

Claim 3.

21. Human embryonic or stem-like cells obtained according to the method of

Claim 4.

22. Human embryonic or stem-like cells obtained according to the method of

Claim 6.

23. Human embryonic or stem-like cells obtained according to the method of

Claim 7.

24. Differentiated human cells obtained by the method of Claim 16.

25. The differentiated human cells of Claim 24, which are selected from the

group consisting of neural cells, hematopoietic cells, pancreatic cells, muscle cells,

cartilage cells, urinary cells, liver cells, spleen cells, reproductive cells, skin cells,

intestinal cells, and stomach cells.

26. A method of therapy which comprises administering to a patient in need of

cell transplantation therapy isogenic differentiated human cells according to Claim 24.

27. The method of Claim 26, wherein said cell transplantation therapy is

effected to treat a disease or condition selected from the group consisting of Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, spinal cord defects or injuries,

multiple sclerosis, muscular dysfrophy, cystic fibrosis, liver disease, diabetes, heart

disease, cartilage defects or injuries, bums, foot ulcers, vascular disease, urinary tract

disease, AIDS and cancer.

28. The method of Claim 26, wherein the differentiated human cells are

hematopoietic cells or neural cells.

29. The method of Claim 26, wherein the therapy is for treatment of Parkinson's

disease and the differentiated cells are neural cells.

30. The method of Claim 26, wherein the therapy is for the treatment of cancer

and the differentiated cells are hematopoietic cells.

31. The differentiated human cells of Claim 24, which contain and express an

inserted gene.

32. The method of Claim 1, wherein a desired gene is inserted, removed or

modified in said embryonic or stem-like cells.

33. The method of Claim 32, wherein the desired gene encodes a therapeutic

enzyme, a growth factor or a cytokine.

34. The method of Claim 32, wherein said embryonic or stem-like cells are

human embryonic or stem-like cells.

35. The method of Claim 32, wherein the desired gene is removed, modified

or deleted by homologous recombination.

36. The method of Claim 1, wherein the donor cell is genetically modified to

impair the development of at least one of endoderm, ectoderm and mesoderm.

37. The method of Claim 1, wherein the donor cell is genetically modified to

increase differentiation efficiency.

38. The method of Claim 36, wherein the cultured nuclear transfer unit is

cultured in a media containing at least one capsase inhibitor.

39. The method of Claim 1 , wherein the donor cell expresses a detectable label

that is indicative of the expression of a particular cyclin.

40. The method of Claim 36, wherein the donor cell has been modified to alter

the expression of a gene selected from the group consisting of SRF, MESP-1, HNF-4,

beta-1, integrin, MSD, GATA-6, GATA-4, RNA helicase A, and H beta 58.

41. The method of Claim 37, wherein said donor cell has been genetically

modified to introduce a DNA that provides for expression of the Q7 and/or Q9 genes.

42. The method of Claim 41, wherein said gene or genes are operably linked

to a regulatable promoter.

43. The method of Claim 1, wherein the donor cell has been genetically

modified to inhibit apoptosis.

44. The method of Claim 43, wherein reduced apoptosis is provided by altering

expression of one or more genes selected from the group consisting of Bad, Bok, BH3,

Bik, Blk, Hrk, BNIP3, Gim_L, Bid, EGL-1, Bcl-XL, Bcl-w, Mcl-l, Al, Nr-13, BHRF-1,

LMW5-HL, ORF16, Ks-Bcl-2, E1B-19K, and CED-9.

45. The method of Claim 44, wherein at least one of said genes is operably

linked to an inducible promoter.

46. A mammalian somatic cell that expresses a DNA that encodes a detectable

marker, the expression of which is linked to a particular cyclin.

47. The cell of Claim 46, wherein the cyclin is selected from the group

consisting of cyclin Dl, D2, D3, Bl, B2, E, A and H.

48. The cell of Claim 46, wherein the detectable marker is a fluorescent

polypeptide.

49. The cell of Claim 48, wherein said mammalian cell is selected from the

group consisting of human, primate, rodent, ungulate, canine, and feline cells.

50. The cell of Claim 48, wherein said cell is a human, bovine or primate cell.