WO2001019977A9 - 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 diagnostic 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. Set, 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 a\., 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 differentiation, 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 demon¬

strating 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 trans¬

fer 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 differ-

entiation in culture of putative pluripotential embryonic cell lines from pig blasto¬

cysts. 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 conditions, the sheep ICMs attach, spread, and

develop areas of both ES cell-like and endoderm-like cells, but that after pro¬

longed 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 HES 1 , 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 attach¬

ment and outgrowth of bovine ICM cells. They concluded based on then 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 serum) 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 propa¬

gation 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 transplantation of bovine ICMs by microinjection 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. Set, 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, previously 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. Set, 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

5 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

degeneration of dopaminergic neurons in the substantia nigra. Standard treatment

for Parkinson's involves administration of L-DOPA, which temporarily

o 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

5 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

o 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 trans-

plantation. 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 thera-

pies.

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 IVP 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 enucleated 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 according 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 embryonic 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 dif¬

ferentiation 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 replacement, burns, vascular diseases, urinary

tract diseases, as well as for the treatment of immune defects, bone marrow trans-

plantation, 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 inven-

tion 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 nu-

cleus 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 matu¬

ration processes during embryonic development which are sufficiently similar or

conserved so as to permit the bovine oocyte to function as an effective substitute

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. Set, 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. Set, 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. Trichostatin 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, "aspira¬

tion" 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 introduced 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 maturation, and most preferably about 16-18 hours after initi¬

ation 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 according to methods known in the art. For

example, the cells may be fused by electrofusion. 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 thermodynamic insta-

bility 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 Inot. 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.

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 ionophore. 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-dimethyl-

amino-purine, staurosporine, 2-aminopurine, and sphingosine.

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 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 eytochalasin

and cycloheximide.

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

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

eytochalasin 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

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

and cycloheximide

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

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

eytochalasin 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.

Natl Acad. Set, 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) fibro¬

blasts, 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 suit¬

es - able 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 PI -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. Set USA, Vol.

95:(23):13709-13713 (1998); andRadice 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 transfer 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., bovines, 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-l, 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 BI 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 embryonic or stem-like cells obtained by the transfer 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

5 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.

o 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.

5 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

o 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. Set, 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 dis-

eases 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 trans¬

plantation. 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 introduce 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 introduced 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, astrocytes have been transfected with BDNF gene

using retroviral 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 tyrosine 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, retroviral

vectors currently used are down-regulated in vivo and the transgene is only

transiently expressed (review by Mulligan, Science, 260:926-932 (1993)). Also,

such studies used primary cells, astrocytes, 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 homolo¬

gous 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 demon-

strated 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), neurotiophin-3, neurotiOphin-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 transfected 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 CR1 aa 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 min 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) supple¬

mented 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, IL),

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 appearance. 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.

I 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 mor¬

phology (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 LIF. 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

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

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

oocytes;

(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

5 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 condi¬

tions 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

0 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

5 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,

o 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 fibro-

blasts.

12. The method of Claim 1, wherein in step (iv) 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 dystrophy, cystic fibrosis, liver

disease, diabetes, heart disease, cartilage defects or injuries, burns, 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

5 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.

o

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.

5

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.

0 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, BNJP3, 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, BI, 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.