Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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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
o This application claims priority under 35 U.S.C. ~119 to PCT/LTS99/0460$,
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.
1 s 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
2 o stem-like cells by transplantation of the nucleus of a primate or human
cell into an
enucleated animal oocyte, e.g., a primate or ungulate oocyte and in a
preferred
embodiment a bovine enucleated oocyte.
The present invention further relates to the use of the resultant embryonic or
stem-
like cells, preferably primate or human embryonic or stem-like cells for
therapy, for diag-
2 5 nostic applications, for the production of differentiated cells which may
also be used for
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therapy or diagnosis, and for the production of transgenic embryonic or
transgenic
differentiated cells, cell lines, tissues and organs. Also, the embryonic or
stem-like cells
obtained according to the present invention may themselves be used as nuclear
donors in
nuclear transplantation or nuclear transfer methods for the production of
chimeras or
clones, preferably transgenic cloned or chimeric animals.
BACKGROUND OF THE INVENTION
Methods for deriving embryonic stem (ES) cell lines in vitro from early
preimplantation mouse embryos are well known. (See, e.g., Evans et al.,
Nature, 29:154-
156 (1981); Martin, Proc. Natl. Acad. Sci., USA, 78:7634-7638 (1981)). ES
cells can be
1 o passaged in an undifferentiated state, provided that a feeder layer of
fibroblast cells
(Evans et al., Id.) or a differentiation inhibiting source (Smith et al., Dev.
Biol., 121:1-9
(1987)) is present.
ES cells have been previously reported to possess numerous applications. For
example, it has been reported that ES cells can be used as an in vitro model
for differen-
tiation, especially for the study of genes which are involved in the
regulation of early
development. Mouse ES cells can give rise to germline chimeras when introduced
into
preimplantation mouse embryos, thus demonstrating their pluripotency (Bradley
et al.,
Nature, 309:255-256 (1984)).
In view of their ability to transfer their genome to the next generation, ES
cells
2 0 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
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animals, e.g., ungulates, nuclei from like preimplantation livestock embryos
support the
development of enucleated oocytes to term (Smith et al., Biol. Reprod.,
40:1027-1035
(1989); and Keefer et al., Biol. Reprod., 50:935-939 (1994)). This is in
contrast to nuclei
from mouse embryos which beyond the eight-cell stage after transfer reportedly
do not
support the development of enucleated oocytes (Cheong et al, Biol. Reprod.,
48:958
(1993)). Therefore, ES cells from livestock animals are highly desirable
because they
may provide a potential source of totipotent donor nuclei, genetically
manipulated or
otherwise, for nuclear transfer procedures.
Some research groups have reported the isolation of purportedly pluripotent
1 o 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., .I. Reprod. Fert. Suppl.,
41:51-56 (1990)
discloses maintenance and differentiation in culture of putative
pluripotential embryonic
cell lines from pig blastocysts. Further, Gerfen et al., Anim. Biotech, 6(1):1-
14 (1995)
disclose the isolation of embryonic cell lines from porcine blastocysts. These
cells are
stably maintained in mouse embryonic fibroblast feeder layers without the use
of
conditioned medium. These cells reportedly differentiate into several
different cell types
2 o during culture (Gerfen et al., Id.).
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Further, Saito et al., Roux's Arch. Dev. Biol., 201:134-141 (1992) report
bovine
embryonic stem cell-like cell lines cultured which survived passages for
three, but were
lost after the fourth passage. Still further, Handyside et al., Roux's Arch.
Dev. Biol.,
196:185-190 (1987) disclose culturing of immunosurgically isolated inner cell
masses of
sheep embryos under conditions which allow for the isolation of mouse ES cell
lines
derived from mouse ICMs. Handyside et al. (1987) (Id.), report that under such
condi-
tions, the sheep ICMs attach, spread, and develop areas of both ES cell-like
and
endoderm-like cells, but that after prolonged culture only endoderm-like cells
are evident.
(Id.)
1o 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
2 o in the mouse. The authors concluded based on their results that ED cells
from day nine
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ovine embryos are totipotent by nuclear transfer and that totipotency is
maintained in
culture.
Van Stekelenburg-Hamers et al., Mol. Reprod. Dev., 40:444-454 (1995), reported
the isolation and characterization of purportedly permanent cell lines from
inner cell mass
cells of bovine blastocysts. The authors isolated and cultured ICMs from 8 or
9 day
bovine blastocysts under different conditions to determine which feeder cells
and culture
media are most efficient in supporting the attachment and outgrowth of bovine
ICM cells.
They concluded based on their results that the attachment and outgrowth of
cultured ICM
cells is enhanced by the use of STO (mouse fibroblast) feeder cells (instead
of bovine
uterus epithelial cells) and by the use of charcoal-stripped serum (rather
than normal se-
rum) to supplement the culture medium. Van Stekelenburg et al reported,
however, that
their cell lines resembled epithelial cells more than pluripotent ICM cells.
(Id.)
Still further, Smith et al., WO 94/24274, published October 27, 1994, Evans et
al,
WO 90/03432, published April 5, 1990, and Wheeler et al, WO 94/26889,
published
November 24, 1994, report the isolation, selection and propagation of animal
stem cells
which purportedly may be used to obtain transgenic animals. Also, Evans et
al.,
WO 90/03432, published on April 5, 1990, reported the derivation of
purportedly
pluripotent embryonic stem cells derived from porcine and bovine species which
assertedly are useful for the production of transgenic animals. Further,
Wheeler et al,
2 o 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
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on the foregoing, it is evident that many groups have attempted to produce ES
cell lines,
e.g., because of their potential application in the production of cloned or
transgenic
embryos and in nuclear transplantation.
The use of ungulate ICM cells for nuclear transplantation has also been
reported.
For example, Collas et al., Mol. Reprod. Dev., 38:264-267 (1994) disclose
nuclear trans-
plantation of bovine ICMs by 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),
1 o disclose the use of bovine ICM cells as donor nuclei in nuclear transfer
procedures, to
produce blastocysts which, upon transplantation into bovine recipients,
resulted in several
live offspring. Further, Sims et al., Proc. Natl. Acad. Sci., USA, 90:6143-
6147 (1993),
disclosed the production of calves by transfer of nuclei from short-term in
vitro cultured
bovine ICM cells into enucleated mature oocytes.
s5 Also, the production of live lambs following nuclear transfer of cultured
embryonic disc cells has been reported (Campbell et al., Nature, 3 80:64-68 (
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).
2 o Further, there have been previous attempts to produce cross species NT
units
(Wolfe et al., Theriogenolo~, 33:350 (1990). Specifically, bovine embryonic
cells were
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fused with bison oocytes to produce some cross species NT units possibly
having an inner
cell mass. However, embryonic cells, not adult cells were used, as donor
nuclei in the
nuclear transfer procedure. The dogma has been that embryonic cells are more
easily
reprogrammed than adult cells. This dates back to earlier NT studies in the
frog (review
by DiBerardino, Differentiation, 17:17-30 (1980)). Also, this study involved
very
phylogenetically similar animals (cattle nuclei and bison oocytes). By
contrast, previ
ously when more diverse species were fused during NT (cattle nuclei into
hamster
oocytes), no inner cell mass structures were obtained. Further, it has never
been
previously reported that the inner cell mass cells from NT units could be used
to form an
1 o 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
2 o through prolonged culture; (iii) maintains the potential to differentiate
into derivatives of
endoderm, mesoderm and ectoderm tissues throughout culture; and (iv) will not
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differentiate when cultured on a fibroblast feeder layer. These cells were
reportedly
negative for the SSEA-1 marker, positive for the SEA-3 marker, positive for
the SSEA-4
marker, express alkaline phosphatase activity, are pluripotent, and have
karyotypes which
include the presence of all the chromosomes characteristic of the primate
species and in
which none of the chromosomes are altered. Further, these cells are
respectfully positive
for the TRA-1-60, and TRA-1-81 markers. The cells purportedly differentiate
into
endoderm, mesoderm and ectoderm cells when injected into a SCID mouse. Also,
purported embryonic stem cell lines derived from human or primate blastocysts
are
discussed in Thomson et al., Science 282:1145-1147 and Proc. Natl. Acad. Sci.,
USA
92:7844-7848 (1995).
Thus, Thomson disclose what purportedly are non-human primate and human
embryonic or stem-like cells and methods for their production. However, there
still exists
a significant need for methods for producing human embryonic or stem-like
cells that are
autologous to an intended transplant recipient given their significant
therapeutic and
diagnostic potential.
In this regard, numerous human diseases have been identified which may be
treated by cell transplantation. For example, Parkinson's disease is caused by
degenera-
tion of dopaminergic neurons in the substantia nigra. Standard treatment for
Parkinson's
involves administration of L-DOPA, which temporarily ameliorates the loss of
dopamine,
2 o 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
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to treatment of many brain diseases and central nervous system injury,
involves
transplantation of cells or tissues from fetal or neonatal animals into the
adult brain. Fetal
neurons from a variety of brain regions can be incorporated into the adult
brain. Such
grafts have been shown to alleviate experimentally induced behavioral
deficits, including
complex cognitive functions, in laboratory animals. Initial test results from
human
clinical trials have also been promising. However, supplies of human fetal
cells or tissue
obtained from miscarriages is very limited. Moreover, obtaining cells or
tissues from
aborted fetuses is highly controversial.
There is currently no available procedure for producing "fetal-like" cells
from the
patient. Further, allograft tissue is not readily available and both allograft
and xenograft
tissue are subject to graft rejection. Moreover, in some cases, it would be
beneficial to
make genetic modifications in cells or tissues before transplantation.
However, many
cells or tissues wherein such modification would be desirable do not divide
well in culture
and most types of genetic transformation require rapidly dividing cells.
There is therefore a clear need in the art for a supply of human embryonic or
stem-
like undifferentiated cells for use in transplants and cell and gene
therapies.
OBJECTS OF THE INVENTION
It is an object of the invention to provide novel and improved methods for
producing embryonic or stem-like cells.
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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 ofthe 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
2 o 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
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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.
l0 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 l, 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
2 o ribozymes specific to genes that induce apoptosis during early embryonic
development.
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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., G 1
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
1 o produced by transplantation of nucleus of an animal or human cell into an
enucleated
oocyte of a different species.
It is a more specific object of the invention to provide primate or human
embryonic or stem-like cells produced by transplantation of the nucleus of a
primate or
human cell into an enucleated animal oocyte, e.g., a human, primate or
ungulate enucle-
ated oocyte.
It is another object of the invention to use such embryonic or stem-like cells
for
therapy or diagnosis.
It is a specific object of the invention to use such primate or human
embryonic or
stem-like cells for treatment or diagnosis of any disease wherein cell, tissue
or organ
2 o transplantation is therapeutically or diagnostically beneficial.
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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 embry-
onic or stem-like cells, which cells may be used to produce genetically
engineered or
z o transgenic differentiated human cells, tissues or organs, e.g., having use
in gene therapies.
It is another specific object of the invention to use the embryonic or stem-
like cells
produced according to the invention in vitro, e.g. for study of cell
differentiation and for
assay purposes, e.g. for drug studies.
It is another object of the invention to provide improved methods of
transplantation therapy, comprising the usage of isogenic or synegenic cells,
tissues or
organs produced from the embryonic or stem-like cells produced according to
the
invention. Such therapies include by way of example treatment of diseases and
injuries
including Parkinson's, Huntington's, Alzheimer's, ALS, spinal cord injuries,
multiple
sclerosis, muscular dystrophy, diabetes, liver diseases, heart disease,
cartilage replace-
2 o ment, burns, vascular diseases, urinary tract diseases, as well as for the
treatment of
immune defects, bone marrow transplantation, cancer, among other diseases.
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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
1 o produced according to the invention for the production of transgenic
animals, e.g., non-
human primates, rodents, ungulates, etc. Such transgenic animals can be used
to produce,
e.g., animal models for study of human diseases, or for the production of
desired
polypeptides, e.g., therapeutics or nutripharmaceuticals.
With the foregoing and other objects, advantages and features of the invention
that
will become hereinafter apparent, the nature of the invention may be more
clearly
understood by reference to the following detailed description of the preferred
embodiments of the invention and to the appended claims.
BRIEFS DESCRIPTION OF THE FIGURES
Figure 1 is a photograph of a nuclear transfer (NT) unit produced by transfer
of an
2 o adult human cell into an enucleated bovine oocyte.
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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
z o 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.
2 o Also, prior to the present invention, no one obtained embryonic or stem-
like cells
by cross-species nuclear transplantation.
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Quite unexpectedly, the present inventors discovered that human embryonic or
stem-like cells and cell colonies may be obtained by transplantation of the
nucleus of a
human cell, e.g., an adult differentiated human cell, into an enucleated
animal oocyte,
which is used to produce nuclear transfer (NT) units, the cells of which upon
culturing
give rise to human embryonic or stem-like cells and cell colonies. This result
is highly
surprising because it is the first demonstration of effective cross-species
nuclear
transplantation involving the introduction of a differentiated donor cell or
nucleus into an
enucleated oocyte of a genetically dissimilar species, e.g., the
transplantation of cell
nuclei from a differentiated animal or human cell, e.g., adult cell, into the
enucleated egg
of a different animal species, to produce nuclear transfer units containing
cells which
when cultured under appropriate conditions give rise to embryonic or stem-like
cells and
cell colonies.
Preferably, the NT units used to produce ES-like cells will be cultured to a
size of
at least 2 to 400 cells, preferably 4 to 128 cells, and most preferably to a
size of at least
about 50 cells.
In the present invention, embryonic or stem-like cells refer to cells produced
according to the present invention. The present application refers to such
cells as stem-
like cells rather than stem cells because of the manner in which they are
typically
produced, i.e., by cross-species nuclear transfer. While these cells are
expected to possess
2 o 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
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may possess the mitochondria of the oocytes used for nuclear transfer, and
thus not
behave identically to conventional embryonic stem cells.
The present discovery was made based on the observation that nuclear
transplantation of the nucleus of an adult human cell, specifically a human
epithelial cell
obtained from the oral cavity of a human donor, when transferred into an
enucleated
bovine oocyte, resulted in the formation of nuclear transfer units, the cells
of which upon
culturing gave rise to human stem-like or embryonic cells and human embryonic
or stem-
like cell colonies. This result has recently been reproduced by
transplantation of
keratinocytes from an adult human into an enucleated bovine oocyte with the
successful
production of a blastocyst and ES cell line. Based thereon, it is hypothesized
by the
present inventors that bovine oocytes and human oocytes, and likely mammals in
general
must undergo maturation processes during embryonic development which are
sufficiently
similar or conserved so as to permit the bovine oocyte to function as an
effective sub-
stitute or surrogate for a human oocyte. Apparently, oocytes in general
comprise factors,
likely proteinaceous or nucleic acid in nature, that induce embryonic
development under
appropriate conditions, and these functions that are the same or very similar
in different
species. These factors may comprise material RNAs andlor 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
2 0 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,
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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
1 o 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 ung.~late 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
2 o nucleus of a donor human cell or a human cell into an enucleated human,
primate, or non-
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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
2 o al, Mol. Report Dev., 38:264-267 (1994); Keefer et al, Biol. Reprod.,
50:935-939 (1994);
Sims et al, Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993); WO 94/26884; WO
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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
1 o used for nuclear transfer may be obtained from different organs, e.g.,
skin, lung, pancreas,
liver, stomach, intestine, heart, reproductive organs, bladder, kidney,
urethra and other
urinary organs, etc. These are just examples of suitable donor cells. Suitable
donor cells,
i.e., cells useful in the subject invention, may be obtained from any cell or
organ of the
body. This includes all somatic or germ cells. Preferably, the donor cells or
nucleus
would comprise actively dividing, i.e., non-quiescent, cells as this has been
reported to
enhance cloning efficacy. Also preferably, such donor cells will be in the Gl
cell cycle.
The resultant blastocysts may be used to obtain embryonic stem cell lines
according to the culturing methods reported by Thomson et al., Science,
282:1145-1147
( 1998) and Thomson et al., Proc. Natl. Acad. Sci., USA, 92:7544-7848 ( 1995),
2 o incorporated by reference in their entirety herein.
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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
1o 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. Bioassays (May, 1995) 17(S): 423-430), as have other
compounds.
2 o For instance, butyrate is also believed to cause hyper-acetylations of
histones by
inhibiting histone deacetylase. Generally, butyrate appears to modify gene
expression and
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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,
to Jaenisch et al. (1997) have reported that 5-azacytidine can be used to
reduce the level of
DNA methylation in cells, potentially leadi_~lg 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
2 0 or ungulates, e.g., a bovine.
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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
1 o slaughterhouse and maturing the oocytes in a maturation medium prior to
fertilization or
enucleation until the oocyte attains the metaphase II stage, which in the case
of bovine
oocytes generally occurs about 18-24 hours post-aspiration. For purposes of
the present
invention, this period of time is known as the "maturation period." As used
herein for
calculation of time periods, "aspiration" refers to aspiration of the immature
oocyte from
ovarian follicles.
Additionally, metaphase II stage oocytes, which have been matured in vivo have
been successfully used in nuclear transfer techniques. Essentially, mature
metaphase II
oocytes are collected surgically from either non-superovulated or
superovulated cows or
heifers 35 to 48 hours past the onset of estrus or past the injection of human
chorionic
2 o gonadotropin (hCG) or similar hormone.
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The stage of maturation of the oocyte at enucleation and nuclear transfer has
been
reported to be significant to the success of NT methods. (See e.g., Prather et
al.,
Differentiation, 48, 1-8, 1991). In general, previous successful mammalian
embryo
cloning practices used metaphase II stage oocyte as the recipient oocyte
because at this
stage it is believed that the oocyte can be or is sufficiently "activated" to
treat the intro-
duced nucleus as it does a fertilizing sperm. In domestic animals, and
especially cattle,
the oocyte activation period generally ranges from about 16-52 hours,
preferably about
28-42 hours post-aspiration.
For example, immature oocytes may be washed in HEPES buffered hamster
1 o 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
2 o 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
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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 CR1 aa, plus 10% estrus cow serum, and then enucleated later,
preferably not
more than 24 hours later, and more preferably 16-18 hours later.
1 o Enucleation may be accomplished microsurgically using a micropipette to
remove
the polar body and the adjacent cytoplasm. The oocytes may then be screened to
identify
those of which have been successfully enucleated. This screening may be
effected by
staining the oocytes with 1 microgram per milliliter 33342 Hoechst dye in
HECM, and
then viewing the oocytes under ultraviolet irradiation for less than 10
seconds. The
oocytes that have been successfully enucleated can then be placed in a
suitable culture
medium.
In the present invention, the recipient oocytes will typically be enucleated
at a time
ranging from about 10 hours to about 40 hours after the initiation of in vitro
maturation,
more preferably from about 16 hours to about 24 hours after initiation of in
vitro matura-
2 o tion, and most preferably about 16-18 hours after initiation of in vitro
maturation.
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Enucleation may be effected before, simultaneous or after nuclear transfer.
Also,
enucleation may be effected before, after or simultaneous to activation.
A single animal or human cell or nucleus derived therefrom which is typically
heterologous to the enucleated oocyte will then be transferred into the
perivitelline space
of the oocyte, typically enucleated, used to produce the NT unit. However,
removal of
endogenous nucleus may alternatively be effected after nuclear transfer. The
animal or
human cell or nucleus and the enucleated oocyte will be used to produce NT
units ac-
cording to methods known in the art. For example, the cells may be fused by
electro-
fusion. Electrofusion is accomplished by providing a pulse of electricity that
is sufficient
1 o to cause a transient break down of the plasma membrane. This breakdown of
the plasma
membrane is very short because the membrane reforms rapidly. Essentially, if
two
adjacent membranes are induced to break down, upon reformation the lipid
bilayers
intermingle and small channels will open between the two cells. Due to the
ther-
modynamic instability of such a small opening, it enlarges until the two cells
become one.
Reference is made to U.S. Patent 4,997,384, by Prather et al., (incorporated
by reference
in its entirety herein) for a further discussion of this process. A variety of
electrofusion
media can be used including e.g., sucrose, mannitol, sorbitol and phosphate
buffered
solution. Fusion can also be accomplished using Sendai virus as a fusogenic
agent
(Graham, Wister Inot. Symp. Monogr., 9, 19, 1969).
2 o 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
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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 ~.m
chamber by application of an electrical pulse of 90-120V for about 15 ,sec,
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
1 o 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
2 o 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
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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 iono-
1 o phore. Other methods of increasing divalent cation levels include the use
of electric
shock, treatment with ethanol and treatment with caged chelators.
Phosphorylation may be reduced by known methods, e.g., by the addition of
kinase
inhibitors, e.g., serine-threonine kinase inhibitors, such as 6-dimethylamino-
purine,
staurosporine, 2-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 ,uM) with 2 mM of DMAP for 4
2 0 minutes;
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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 ,uM) with 2 mM of DMAP for four
minutes;
2- Move the oocytes into culture media with 2 mM of DMAP and 200
microM of Roscovitin for three hours;
3- Rinse four times and place in culture.
3. Activation by exposure to Ionomycin followed by cytochalasin and
cycloheximide.
1- Place oocytes in Ionomycin (5 microM) for four minutes;
2- Move oocytes to culture media containing 5 ,ug/ml of cytochalasin
B and 5 ,ug/ml of cycloheximide for five hours;
z 5 3- Rinse four times and place in culture.
4. Activation by electrical pulses
1- Place eggs in mannitol media containing 100 ,uM CaCL2;
2- Deliver three pulses of 1.0 kVcrri I for 20 ,usec, each pulse 22
minutes apart;
2 0 3- Move oocytes to culture media containing 5 ~cg/ml of cytochalasin
B for three hours.
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5. Activation by exposure with ethanol followed by cytochalasin and
cycloheximide
1- Place oocytes in 7% ethanol for one minute;
2- Move oocytes to culture media containing 5 ~cg/ml of cytochalasin
B and 5 ~cg/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
,uM 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
2 o Place oocytes or NT units, typically about 22 to 28 hours post
maturation in about 2 mM DMAP for about one hour, followed by
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incubation for about two to twelve hours, preferably about eight hours, in
~sg/ml of cytochalasin B and 20 ,ug/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.
5 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
1 o 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
2 o 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
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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 ~.g/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
1 o 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 CRI,
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
2 o thereon.
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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. Sci.,
USA, 92:7844-7848 (1995).
Afterward, the cultured NT unit or units are preferably washed and then placed
in
a suitable media, e.g., CRIaa medium, Ham's F-10, Tissue Culture Media -199
(TCM-
199). Tyrodes-Albumin-Lactate-Pyruvate ( TALP) Dulbecco's Phosphate Buffered
Saline
(PBS), Eagle's or Whitten's, preferably containing about 10% FCS. Such
culturing will
preferably be effected in well plates which contain a suitable confluent
feeder layer.
Suitable feeder layers include, by way of example, fibroblasts and epithelial
cells, e.g.,
1 o fibroblasts and uterine epithelial cells derived from ungulates, chicken
fibroblasts, murine
(e.g., mouse or rat) fibroblasts, STO and SI-m220 feeder cell lines, and BRL
cells.
In the preferred embodiment, the feeder cells will comprise mouse embryonic
fibroblasts. Means for preparation of a suitable fibroblast feeder layer are
described in
the example which follows and is well within the skill of the ordinary
artisan.
The NT units are cultured on the feeder layer until the NT units reach a size
suitable for obtaining cells which may be used to produce embryonic stem-like
cells or
cell colonies. Preferably, these NT units will be cultured until they reach a
size of at least
about 2 to 400 cells, more preferably about 4 to 128 cells, and most
preferably at least
about 50 cells. The culturing will be effected under suitable conditions,
i.e., about 38.5 °C
2 o and 5% CO2, with the culture medium changed in order to optimize growth
typically
about every 2-5 days, preferably about every 3 days.
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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
1 o 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 P1-catalog
#99242
medium, and the P-1 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
2 o 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
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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.
1 o 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
2 0 or more mesoderm, endoderm or ectoderm specific genes. Examples thereof
include:
Mesoderm: SRF, MESP-1, HNF-4, beta-I integrin, MSD;
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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(1-2):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);
1o Morrisey et al, Genes, Dev., Vol. 12(22):3579-3590 (1998); Soudais et al,
Development,
Vol. 121(11):3877-3888 (1995) (endoderm); and Lee et al, Proc. Natl. Acad.
Sci. USA,
Vol. 95:(23):13709-13713 (1998); and Radice et al, Development, Vol.
111(3):801-811
(1991) (ectoderm).
In general, a desired somatic cell, e.g., a human keratinocyte, epithelial
cell or
fibroblast, will be genetically engineered such that one or more genes
specific to
particular cell lineages are "knocked out" and/or the expression of such genes
significantly impaired. This may be effected by known methods, e.g.,
homologous
recombination. A preferred genetic system for effecting "knock-out" of desired
genes is
disclosed by Capecchi et al, U.S. Patents 5,631,153 and 5,464,764, which
reports
2 o positive-negative selection (PNS) vectors that enable targeted
modification of DNA
sequences in a desired mammalian genome. Such genetic modification will result
in a
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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
1 o 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
2 o ribozymes, or by gene knockout. These "mortal" cells, when used for
nuclear transfer,
will not be capable of differentiating into viable offspring.
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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 andlor 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.
1 o 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.
2 o 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
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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
1 o 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, BimL, Bad, Bid, and EGL-1. By
contrast,
genes that reportedly protect cells from programmed cell death include, by way
of
example, BcL-XL, Bcl-w, Mcl-1, Al, Nr-13, BHRF-1, LMWS-HL, ORF16, Ks-Bel-2,
E 1 B-19K, and CED-9.
Thus, donor cells can be constructed wherein genes that induce apoptosis are
2 0 "knocked out" or wherein the expression of genes that protect the cells
from apoptosis is
enhanced or turned on during embryonic development.
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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.
1 o 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 l,
and
capsases. Additionally, a transgene may be introduced that encodes for
methylase or
2 o 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
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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).
1 o 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 D 1 gene in order to select for cells that are in G 1.
However, any
cyclin gene should be suitable for use in the claimed invention. (See, e.g.,
King et al,
2 o Mol. Biol. Cell, Vol. 7(9):1343-1357 (1996)).
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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 D1, D2 and D3 in G1 phase, cyclin B1 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
1 o 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 D 1 gene which can be used to select
for cells that
2 o are in G1. 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)).
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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
1 o 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
2 o preferably 0.01 % to 1.0% by weight of medium. Thus, the foregoing methods
may be
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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
1 o 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
2 o L-glutamine. The growth medium is changed as often as necessary to
optimize growth,
e.g., about every 2-3 days.
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This culturing process results in the formation of embryonic or stem-like
cells or
cell lines. In the case of human cell/bovine oocyte derived NT embryos,
colonies are
observed by about the second day of culturing in the alpha MEM medium.
However, this
time may vary dependent upon the particular nuclear donor cell, specific
oocyte and
culturing conditions. One skilled in the art can vary the culturing conditions
as desired
to optimize growth of the particular embryonic or stem-like cells. Other
suitable media
are disclosed herein.
The embryonic or stem-like cells and cell colonies obtained will typically
exhibit
an appearance similar to embryonic or stem-like cells of the species used as
the nuclear
cell donor rather than the species of the donor oocyte. For example, in the
case of embry-
onic or stem-like cells obtained by the 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,
2 o that primate stem cells are SSEA-1 (-), SSEA-4 (+), TRA-1-60 (+), TRA-1-81
(+) and
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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,
1 o 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 mitochondria)
DNA of the
2 o same species as the donor cell or nucleus into the recipient oocyte before
or after nuclear
transfer, before or after activation, and before or after fusion and cleavage.
Preferably,
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if the donor cell is human, human mitochondria) DNA will be derived from cells
of the
particular donor, e.g., liver cells and tissue.
Methods for isolating mitochondria are well known in the art. Mitochondria can
be isolated from cells in tissue culture, or from tissue. The particular cells
or tissue will
depend upon the particular species of the donor cell. Examples of cells or
tissues that
may be used as sources of mitochondria include fibroblasts, epithelium, liver,
lung,
keratinocyte, stomach, heart, bladder, pancreas, esophageal, lymphocytes,
monocytes,
mononuclear cells, cumulus cells, uterine cells, placental cells, intestinal
cells,
hematopoietic cells, and tissues containing such cells.
1 o For example, mitochondria can be isolated from tissue culture cells and
rat liver.
It is anticipated that the same or similar procedures may be used to isolate
mitochondria
from other cells and tissues. As noted above, preferred source of mitochondria
comprises
human liver tissue because such cells contain a large number of mitochondria.
Those
skilled in the art will be able to modify the procedure as necessary,
dependent upon the
particular cell line or tissue. The isolated DNA can also be further purified,
if desired,
known methods, e.g., density gradient centrifugation.
In this regard, it is known that mouse embryonic stem (ES) cells are capable
of
differentiating into almost any cell type, e.g., hematopoietic stem cells.
Therefore, human
embryonic or stem-like cells produced according to the invention should
possess similar
2 o 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.
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For example, the subject human embryonic or stem-like cells may be induced to
differentiate into hematopoietic stem cells, muscle cells, cardiac muscle
cells, liver cells,
cartilage cells, epithelial cells, urinary tract cells, etc., by culturing
such cells in
differentiation medium and under conditions which provide for cell
differentiation.
Medium and methods which result in the differentiation of embryonic stem cells
are
known in the art as are suitable culturing conditions.
For example, Palacios et al, Proc. Natl. Acad. Sci., USA, 92:7530-7537 (1995)
teaches the production of hematopoietic stem cells from an embryonic cell line
by
subjecting stem cells to an induction procedure comprising initially culturing
aggregates
of such cells in a suspension culture medium lacking retinoic acid followed by
culturing
in the same medium containing retinoic acid, followed by transferral of cell
aggregates
to a substrate which provides for cell attachment.
Moreover, Pedersen, J. Reprod. Fertil. Dev., 6:543-552 (1994) is a review
article
which references numerous articles disclosing methods for in vitro
differentiation of
embryonic stem cells to produce various differentiated cell types including
hematopoietic
cells, muscle, cardiac muscle, nerve cells, among others.
Further, Bain et al, Dev. Biol., 168:342-357 (1995) teaches in vitro
differentiation
of embryonic stem cells to produce neural cells which possess neuronal
properties. These
references are exemplary of reported methods for obtaining differentiated
cells from
2 o embryonic or stem-like cells. These references and in particular the
disclosures therein
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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.,
s 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
1 o 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
1 s 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
2 o embryonic or stem-like cells as described above, and culturing such cells
under conditions
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which favor differentiation, until hematopoietic stem cells are obtained. Such
hematopoietic cells may be used in the treatment of diseases including cancer
and AIDS.
Alternatively, adult somatic cells from a patient with a neurological disorder
may
be fused with an enucleated animal oocyte, e.g., a primate or bovine oocyte,
human
embryonic or stem-like cells obtained therefrom, and such cells cultured under
differentiation conditions to produce neural cell lines. Specific diseases
treatable by
transplantation of such human neural cells include, by way of example,
Parkinson's
disease, Alzheimer's disease, ALS and cerebral palsy, among others. In the
specific case
of Parkinson's disease, it has been demonstrated that transplanted fetal brain
neural cells
1 o make the proper connections with surrounding cells and produce dopamine.
This can
result in long-term reversal of Parkinson's disease symptoms.
To allow for specific selection of differentiated cells, donor cells may be
transfected with selectable markers expressed via inducible promoters, thereby
permitting
selection or enrichment of particular cell lineages when differentiation is
induced. For
example, CD34-neo may be used for selection of hematopoietic cells, Pwl-neo
for
muscle cells, Mash-1-neo for sympathetic neurons, Mal-neo for human CNS
neurons of
the grey matter of the cerebral cortex, etc.
The great advantage of the subject invention is that it provides an
essentially
limitless supply of isogenic or synegenic human cells suitable for
transplantation.
2 o 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-
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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
1 o 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
2 o genome.
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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
1 o 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
2 o 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.
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Moreover, it is almost impossible to propagate a large population of gene
targeted
primary cells to be used in homologous recombination techniques.
By contrast, the difficulties associated with retroviral systems should be
eliminated
by the use of human embryonic or stem-like cells. It has been demonstrated
previously
by the subject assignee that cattle and pig embryonic cell lines can be
transfected and
selected for stable integration of heterologous DNA. Such methods are
described in
commonly assigned U.S. Serial No. 08/626,054, filed April 1, 1996, now U.S.
Patent No.
5,905,042, incorporated by reference in its entirety. Therefore, using such
methods or
other known methods, desired genes may be introduced into the subj ect human
embryonic
or stem-like cells, and the cells differentiated into desired cell types,
e.g., hematopoietic
cells, neural cells, pancreatic cells, cartilage cells, etc.
Genes which may be introduced into the subject embryonic or stem-like cells
include, by way of example, epidermal growth factor, basic fibroblast growth
factor, glial
derived neurotrophic growth factor, insulin-like growth factor (I and II),
neurotrophin-3,
neurotrophin-4/5, ciliary neurotrophic factor, AFT-1, cytokine genes
(interleukins,
interferons, colony stimulating factors, tumor necrosis factors (alpha and
beta), etc.),
genes encoding therapeutic enzymes, collagen, human serum albumin, etc.
In addition, it is also possible to use one of the negative selection systems
now
known in the art for eliminating therapeutic cells from a patient if
necessary. For
2 o 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
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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 subj ect 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.
to 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,
2 0 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
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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
1 o 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
15 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 ug/ml; Sigma). Individual donor cells were then placed into the
perivitelline
2 o 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
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90 V for 15 .sec was applied to the NT unit. This occurred at 24 hours post-
initiation of
maturation (hpm) of the oocytes. The NT units were placed in CRlaa medium
until 28
hpm.
The procedure used to artificially activate oocytes has been described
elsewhere.
NT unit activation was at 28 hpm. A brief description of the activation
procedure is as
follows: NT units were exposed for four min to ionomycin (5 ~,M; 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% COZ 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% CO2. 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 laXer
Primary cultures of embryonic fibroblasts were obtained from 14-16 day old
2 o 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
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EDTA solution (0.05% trypsin/0.02% EDTA; GIBCO, Grand Island, NY). Fibroblast
cells were plated in tissue culture flasks and cultured in alpha-MEM medium
(BioWhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (FCS)
(Hyclone, Logen, UT), penicillin (100 IU/ml) and streptomycin (SO ~,1/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% COz 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.
1 o NT unit cells obtained as described above were washed and plated directly
onto
irradiated feeder fibroblast cells. These cells included those of the inner
portion of the
NT unit. The cells were maintained in a growth medium consisting of alpha MEM
supplemented with 10% FCS and 0.1 mM beta-mercaptoethanol (Sigma). Growth
medium was exchanged every two to three days. The initial colony was observed
by the
second day of culture. The colony was propagated and exhibits a similar
morphology to
previously disclosed mouse embryonic stem (ES) cells. Individual cells within
the colony
are not well defined and the perimeter of the colony is refractile and smooth
in ap-
pearance. The cell colony appears to have a slower cell doubling time than
mouse ES
cells. Also, unlike bovine and porcine derived ES cells, the colony does not
have an
2 o epithelial appearance thus far. Figures 2 through 5 are photographs of ES-
like cell
colonies obtained as described, supra.
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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.
RE T
Table 1. Human cells as donor nuclei in NT unit production and development.
TABLE 1
Cell type No. NT No. NT unitsNo. NT units No. NT units
2 to 4 to
units madecell stage - 16 cell stage16 - 400 cell
(%) (%)
stage (%)
lymphocytes 18 12 (67%) 3 (17%) 0
oral cavity 34 18 (53%) 3 (9%) 1 (3%)
epithelium
adult fibro- 12 (4 cell;
26%)
blasts 46 4 (9%) 8 (8-16 cells; ---
17.4%)
The one NT unit that developed a structure having greater than 16 cells was
plated
down onto a fibroblast feeder layer. This structure was attached to the feeder
layer and
started to propagate forming a colony with a ES cell-like morphology (See,
e.g., Figure
2). Moreover, although the 4 to 16 cell stage structures were not used to try
and produce
an ES cell colony, it has been previously shown that this stage is capable of
producing ES
or ES-like cell lines (mouse, Eistetter et al., Devel. Growth and Differ.,
31:275-282
(1989); Bovine, Stice et al., 1996)). Therefore, it is expected that 4 - 16
cell stage NT
units should also give rise to embryonic or stem-like cells and cell colonies.
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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 ILT 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
1 o 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 homogenizes
using a
tight-fitting pestle, and the mitochondria are isolated by differential
centrifugation.
The solutions, tubes, and homogenizes 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 homogenizes.
RSB Buffer
RSB (A hypotonic buffer for swelling the tissue culture cells)
2 0 10 mM NaCI
1.5 mM MgCl2
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mM Tris-HCI, pH 7.5
MgCl2
The cells are allowed to swell for five to ten minutes. The progress of the
swelling
is maintained using a phase contrast microscope. The swollen cells are
replaced,
5 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
10 175 Mm sucrose
12.5 mM ris-HCI, pH 7.5
215 mM EDTA pH 7.5
lx MS Buffer
210 mM mannitol
70 mM Sucrose
5 mM Tris-HCI, pH 7.5
1 mM EDTA, pH 7.5
MS Buffer is an iso-osmotic buffer to maintain the tonicity of the
organelles and prevent agglutination.
2 o 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
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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 1X 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
1 o preferably will be used shortly thereafter for NT.
This basic protocol can be modified. In particular, it may be desirable to
further
isolate mitochondria) 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 mitochondria) pellet is omitted as is
the
density gradient purification. Instead, the mitochondria) pellet is simply
resuspended and
lysed, and the mitochondria) DNA purified from any remaining nuclear DNA. As
noted,
suitable methods for purifying mitachondria and mitochondria) DNA are well
known in
2 o the art.
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Homogenization works best if the cells are resuspended in at least 5-lOX the
volume of the cell pellet and if the cell suspension fills the homogenizes at
least half full.
Press the homogenizes pestle straight down the tube, maintaining a firm,
steady pressure.
The Dounce homogenizes 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 mitochondria) isolation protocol is selected based on the particular tissue.
For
1 o 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 mitochondria) 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 homogenizes can be used homogenize rat liver. Alternatively, if the
tissue is
soft enough, a Dounce homogenizes with a loose pestle can be used. The yield
and purity
of the mitochondria) 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.
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Preferably, the buffer, tubes, and homogenizes will be pre-chilled. Pre-
chilling a
glass and Teflon type homogenizes 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 (1X MS) to remove most of the
blood, and
the tissue transferred to the homogenizes tube. Enough homogenization buffer
if added
1 o 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;
2 0 (ii) non-activated, enucleated bovine oocytes;
(iii) activated, enucleated bovine oocytes;
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(iv) non-activated, fused (with human donor cell nr nucleucl hnvinP nnwtPC~
(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 mitochondria) suspension are injected into any of the above.
The
introduction of such mitochondria will result in NT units wherein the
rnitochondrial and
1 o 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)
2 o embryo;
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(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
1 o 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 (>10') 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
2 o enucleation procedure by the sterile addition of 30 ml DMEM, 2 ml bovine
serum, and
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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
1 o 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.
2 0 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
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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 COZ 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
1 o 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.
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