Note: Descriptions are shown in the official language in which they were submitted.
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CLONING USING DONOR NUCLEI ROM
NON-SERUM STARED DIFFERENTIATED CELLS
1. FIELD OF THE INVENTION
The present invention relates to cloning procedures in which cell
nuclei derived from non-serum starved, differentiated, mammalian cells are
transplanted into enucleated mammalian oocytes of the same species as the
donor nuclei. The nuclei are reprogrammed to direct the development of
cloned embryos, which can then be transferred into recipient females to
produce fetuses and offspring, or used to produce cultured inner cell mass
cells (C1CM). The cloned embryos can also be combined with fertilized
embryos to produce chimeric embryos, fetuses and/or offspring.
2. BACKGROUND OF THE INVENTION
The use of ungulate inner cell mass (ICM) cells for nuclear
transplantation has been reported. For example, Collas et al., Mol. Reprod.
Dev., 38:264-267 (1994) discloses nuclear transplantation of bovine ICMs
by microinjection of the lysed donor cells into enucleated mature oocytes.
Collas et af. 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), disclosed 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.
The production of five Iambs following nuclear transfer of cultured
embryonic disc cells has also been reported (Campbell et al., Nature,
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380:64-68 (1996)). Still further, the use of bovine pluripotent embryonic
cells in nuclear transfer and the production of chimeric fetuses has been
reported (Stice et al., Biol. Reprod., 54:100-110 (1996); Collas et al, Mol.
Reprod. Dev., 38:264-267 (1994)). Collas et al demonstrated that
granulosa cells (adult cells) could be used in a bovine cloning procedure to
produce embryos. However, there was no demonstration of development
past early embryonic stages (biastocyst stage). Also, granulosa cells are
not easily cultured and are only obtainable from females. Collas et al did
not attempt to propagate the granulosa cells in culture or try to genetically
to modify those cells. Wilmut et al (Nature, 355:810-813 (1997)) produced
nuclear transfer sheep offspring derived from fetal fibroblast cells, and one
offspring from a cell derived from an adult sheep.
There also exist problems in the area of producing transgenic
mammals. By current methods, heterologous DNA is introduced into either
early embryos or embryonic cell lines that differentiate into various cell
types in the fetus and eventually develop into a transgenic animal.
However, many early embryos are required to produce one transgenic
animal and, thus, this procedure is very inefficient. Also, there is no simple
and efficient method of selecting for a transgenic embryo before going
2 o through the time and expense of putting the embryos into surrogate
females. In addition, gene targeting techniques cannot be easily
accomplished with early embryo transgenic procedures.
Embryonic stem cells in mice have enabled researchers to select for
transgenic cells and perform gene targeting. This allows more genetic
engineering than is possible with other transgenic techniques. However,
embryonic stem cell lines and other embryonic cell lines must be maintained
in an undifferentiated state that requires feeder layers and/or the addition
of
cytokines to media. Even if these precautions are followed, these cells
often undergo spontaneous differentiation and cannot be used to produce
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transgenic offspring by currently available methods. Also, some embryonic
cell lines have to be propagated in a way that is not conducive to gene
targeting procedures.
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 )1. ES cells can be passaged in an undifferentiated
state, provided that a feeder layer of fibroblast cells (Evans et al., id.) or
a
differentiation inhibiting source (Smith et al., Dev. Biol., 127:1-9 (1987))
is
l0 present.
ES cells have been previously reported to possess numerous
applications. For example, it has been reported that ES cells can be used as
an in vitro model for differentiation, especially for the study of genes which
are involved in the regulation of early development. Mouse ES cells can
give rise to germline chimeras when introduced into preimplantation mouse
embryos, thus demonstrating their pluripotency (Bradley et al., Nature,
309:255-256 (1984)).
In view of their ability to transfer their genome to the next
generation, ES cells have potential utility for germfine manipulation of
livestock animals by using ES cells with or without a desired genetic
modification. Moreover, in the case of livestock animals, e.g., ungulates,
nuclei from like preimplantation livestock embryos support the development
of enucleated oocytes to term (Smith et al., Biol. Reprod., 40:1027-1035
(1989); and Keefer et al., Biol. Reprod., 50:935-939 (1994)). This is in
2 5 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
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otherwise, for nuclear transfer procedures.
Some research groups have reported the isolation of purportedly
pluripotent embryonic cell lines. For example, Notarianni et al., J. Reprod.
Fert. Suppl., 43:255-260 (1991 ), reports 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. Also, Notarianni et al., J. Reprod. Fert. Suppl., 41:51-56
(1990) discloses maintenance and differentiation in culture of putative
1c5 pluripotential embryonic cell lines from pig blastocysts. Gerfen et al.,
Anim.
Biotech, 6(1 ):1-14 (1995) discloses the isolation of embryonic cell lines
from porcine blastocysts. These cells are stably maintained in mouse
embryonic fibroblast feeder layers without the use of conditioned medium,
and reportedly differentiate into several different cell types during culture.
Further, Saito et al., Roux's Arch. Dev. Biol., 201:134-141 (1992)
reports cultured, bovine embryonic stem cell-like cell lines which survived
three passages, but were lost after the fourth passage. Handyside et al.,
Roux's Arch. Dev. Biol., 196:185-190 (1987) discloses culturing of
immunosurgically isolated inner cell masses of sheep embryos under
2 o conditions which allow for the isolation of mouse ES cell lines derived
from
mouse ICMs. Handyside et al. reports that under such conditions, 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.
2 5 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 stained positive for alkaline
phosphatase (AP), exhibited the ability to form embryoid bodies, and
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spontaneously differentiated into at least two different cell types. These
cells also reportedly expressed mRNA for the transcription factors OCT4,
OCT6 and HES1, a pattern of homeobox genes which is believed to be
expressed by ES cells exclusively.
Also recently, Campbell et al., Nature, 380:64-68 ( 1996) reported
the production of live Iambs following nuclear transfer of cultured
embryonic disc (ED) cells from day nine ovine embryos cultured under
conditions which promote the isolation of ES cell lines in the mouse. The
authors concluded that ED cells from day nine ovine embryos are totipotent
1o 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
s5 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 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
20 (rather than normal serum) to supplement the culture medium. Van
Stekelenburg et al reported, however, that their cell lines resembled
epithelial cells more than pluripotent ICM cells.
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
25 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. Evans et al. also reported the derivation of
purportedly pluripotent embryonic stem cells from porcine and bovine
species which assertedly are useful for the production of transgenic
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animals. Further, Wheeler et al, WO 94/26884, published November 24,
1994, disclosed embryonic stem cells which are assertedly useful for the
manufacture of chimeric and transgenic ungulates.
Thus, based on the foregoing, it is evident that many groups have
attempted to produce ES cell lines, e.g., because of their potential
application in the production of cloned or transgenic embryos and in nuclear
transplantation.
Notwithstanding what has previously been reported in the literature,
there exists a need for improved methods of cloning mammalian cells.
OBJECTS AND SUMMARY OF THE INVENTION
It is an object of the invention to provide novel and improved
methods for producing cloned mammals (e.g., embryos, fetuses and
offspring).
It is a more specific object of the invention to provide a novel method
for cloning mammals which involves transplantation of the nucleus of a
non-serum starved, differentiated mammalian cell into an enucleated oocyte
of the same species.
2 o It is another object of the invention to provide a method for
multiplying adult mammals having proven genetic superiority or other
desirable traits.
It is another object of the invention to provide an improved method
for producing genetically engineered or transgenic mammals (i.e., embryos,
fetuses, offspring). The invention also provides genetically engineered or
transgenic mammals made by such a method.
It is a more specific object of the invention to provide a method for
producing genetically engineered or transgenic mammals by which a desired
DNA sequence is inserted, removed or modified in a differentiated
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mammalian cell or cell nucleus prior to use of that differentiated cell or
cell
nucleus for formation of a NT unit. The invention also provides genetically
engineered or transgenic mammals made by such a method.
It is another object of the invention to provide a method for
producing genetically engineered or transgenic mammals by transplantation
of the nucleus of a non-serum starved, transgenic, differentiated cell into an
enucleated oocyte of the same species as the differentiated cell. The
invention also provides genetically engineered or transgenic mammals made
by such a method.
It is another object of the invention to provide a novel method for
producing mammalian CICM cells which involves transplantation of a
nucleus of a non-serum starved, differentiated cell into an enucleated
oocyte of the same species as the differentiated cell.
It is another object of the invention to provide CICM cells produced
by transplantation of the nucleus of a non-serum starved, differentiated
mammalian cell into an enucleated oocyte of the same species as the
differentiated cell.
It is a more specific object of the invention to provide a method for
producing human C1CM cells which involves transplantation of nuclei of a
2 o non-serum starved, human cell, e.g., a human adult cell, into an
enucleated
human oocyte.
It is another object of the invention to use such CICM cells for
therapy or diagnosis.
It is a specific object of the invention to use such CICM cells,
including human and ungulate CICM cells, for treatment or diagnosis of any
disease wherein cell, tissue or organ transplantation is therapeutically or
diagnostically beneficial. The CICM cells may be used within the same
species or across species.
tt is another object of the invention to use cells or tissues derived
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from NT embryos, fetuses or offspring, including human and ungulate
tissues, for treatment or diagnosis of any disease or injury wherein cell,
tissue or organ transplantation is therapeutically or diagnostically
beneficial.
Such diseases and injuries include Parkinson's, Huntington's, Alzheimer's,
ALS, spinal cord injuries, multiple sclerosis, muscular dystrophy, diabetes,
liver diseases, heart disease, cartilage replacement, burns, vascular
diseases, urinary tract diseases, as well as for the treatment of immune
defects, bone marrow transplantation, cancer, among other diseases.
The tissues may be used within the same species or across species.
o It is another specific object of the invention to use the CICM 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 human CICM
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 CICM 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
2 o transplantation therapy, comprising the usage of isogenic or syngenic
cells,
tissues or organs produced from the CICM cells produced according to the
invention. Such therapies include by way of example treatment of diseases
and injuries including Parkinson's, Huntington's, Alzheimer's, ALS, spinal
cord injuries, multiple sclerosis, muscular dystrophy, diabetes, liver
diseases, heart disease, cartilage replacement, burns, vascular diseases,
urinary tract diseases, as well as for the treatment of immune defects, bone
marrow transplantation, cancer, among other diseases.
It is another object of the invention to provide genetically engineered
or transgenic CICM cells produced by inserting, removing or modifying a
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desired DNA sequence in a differentiated mammalian cell or cell nucleus
prior to use of that differentiated cell or cell nucleus for formation of a NT
unit.
It is another object of the invention to use the transgenic or
genetically engineered CICM 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 CICM cells produced
according to the invention or transgenic or genetically engineered CICM
cells produced according to the invention as nuclear donors for nuclear
transplantation.
Thus, in one aspect, the present invention provides a method for
cloning a mammal (e.g., embryos, fetuses, offspring). The method
comprises:
(i) inserting a desired non-serum starved, differentiated
mammalian cell or cell nucleus into an enucleated mammalian oocyte of the
same species as the differentiated cell or cell nucleus, under conditions
suitable for the formation of a nuclear transfer (NT) unit;
(ii) activating the resultant nuclear transfer unit; and
2 o (iii) transferring said cultured NT unit to a host mammal such that the
NT unit develops into a fetus.
Preferably, the activated nuclear transfer unit is cultured until greater
than the 2-cell developmental stage.
The cells, tissues and/or organs of the fetus are advantageously used
in the area of cell, tissue and/or organ transplantation.
The present invention also includes a method of cloning a genetically
engineered or transgenic mammal, by which a desired DNA sequence is
inserted, removed or modified in the differentiated mammalian cell or cell
nucleus prior to insertion of the differentiated mammalian cell or cell
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nucleus into the enucleated oocyte.
Also provided by the present invention are mammals obtained
according to the above method, and offspring of those mammals.
The present invention is preferably used for cloning ungulates.
In another aspect, the present invention provides a method for
producing CICM cells. The method comprises:
(i) inserting a desired non-serum starved, differentiated
mammalian cell or cell nucleus into an enucleated mammalian oocyte of the
same species as the differentiated cell or cell nucleus, under conditions
1o suitable for the formation of a nuclear transfer (NT) unit;
(ii) activating the resultant nuclear transfer unit; and
(iii) culturing cells obtained from said cultured NT unit to obtain CICM
cells.
Preferably, the activated nuclear transfer unit is cultured until greater
than the 2-cell developmental stage.
The CICM cells are advantageously used in the area of cell, tissue
and organ transplantation.
With the foregoing and other objects, advantages and features of the
invention that will become hereinafter apparent, the nature of the invention
2 o may be more clearly understood by reference to the following detailed
description of the preferred embodiments of the invention and to the
appended claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides improved procedures for cloning
mammals by nuclear transfer or nuclear transplantation. In the subject
application, nuclear transfer or nuclear transplantation or NT are used
interchangeably.
According to the invention, cell nuclei derived from non-serum
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starved, differentiated mammalian cells are transplanted into enucleated
mammalian oocytes of the same species as the donor nuclei. The nuclei
are reprogrammed to direct the development of cloned embryos, which can
then be transferred into recipient females to produce fetuses and offspring,
or used to produce CICM cells. The cloned embryos can also be combined
with fertilized embryos to produce chimeric embryos, fetuses and/or
offspring.
Prior art methods have used embryonic cell types in cloning
procedures. This includes work by Campbell et al (Nature, 380:64-68,
1996) and Stice et al (Biol. Reprod., 54:100-1 10, 1996). In both of those
studies, embryonic cell lines were derived from embryos of less than 10
days of gestation. In both studies, the cells were maintained on a feeder
layer to prevent overt differentiation of the donor cell to be used in the
cloning procedure. The present invention has been found to be effective
using either fetal or adult cells.
it was unexpected that cloned embryos with fetal or adult donor
nuclei could develop to advanced embryonic and fetal stages. The
scientific dogma has been that only early embryonic cell types could direct
this type of development. It was unexpected that a large number of cloned
2 o embryos could be produced from fetal or adult cells. Also, the fact that
new transgenic embryonic cell lines could be readily derived from transgenic
cloned embryos was unexpected.
Adult cells and fetal fibroblast cells from a sheep have purportedly
been used to produce a sheep offspring (Wilmut et al, 1997). In that
study, however, it was emphasized that the use of a serum starved,
nucleus donor cell in the quiescent state was important for success of the
Wilmut cloning method. No such requirement for serum starvation or
quiescence exists for the present invention. On the contrary, cloning is
achieved using non-serum starved, differentiated mammalian cells.
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Moreover, cloning efficiency according to the present invention can be the
same regardless of whether fetal or adult donor cells are used, whereas
Wilmut et al ( 1997) reported that lower cloning efficiency was achieved
with adult donor cells.
Thus, according to the present invention, multiplication of superior
genotypes of mammals, including ungulates, is possible. This will allow the
multiplication of adult animals with proven genetic superiority or other
desirable traits. Progress will be accelerated, for example, in many
important ungulate species. By the present invention, there are potentially
1o billions of fetal or adult cells that can be harvested and used in the
cloning
procedure. This will potentially result in many identical offspring in a short
period.
There has also been speculation that the Wilmut et al method will
lead to the generation of transgenic animals (see MacQuitty, Nature
Biotech., 15:294 (1997)). However, there is no reason to assume, for
example, that nuclei from adult cells that have been transfected with
exogenous DNA will be able to survive the process of nuclear transfer. In
this regard, it is known that the properties of mouse embryonic stem (ES)
cells are altered by in vitro manipulation such that their ability to form
viable
2o chimeric embryos is effected. Therefore, prior to the present invention,
the
cloning of transgenic animals could not have been predicted.
The present invention allows simplification of transgenic procedures
by working with a cell source that can be clonally propagated. This
eliminates the need to maintain the cells in an undifferentiated state, thus,
genetic modifications, both random integration and gene targeting, are more
easily accomplished. Also by combining nuclear transfer with the ability to
modify and select for these cells in vitro, this procedure is more efficient
than previous transgenic embryo techniques. According to the present
invention, these cetls can be ctonally propagated without cytokines,
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conditioned media and/or feeder layers, further simplifying and facilitating
the transgenic procedure. When transfected cells are used in cloning
procedures according to the invention, transgenic embryos are produced
which can develop into fetuses and offspring. Also, these transgenic
cloned embryos can be used to produce CICM cell lines or other embryonic
cell lines. Therefore, the present invention eliminates the need to derive
and maintain in vitro an undifferentiated cell line that is conducive to
genetic engineering techniques.
The present invention can also be used to produce CICM cells,
10- fetuses or offspring which can be used, for example, in cell, tissue and
organ transplantation. By taking a fetal or adult cell from an animal and
using it in the cloning procedure a variety of cells, tissues and possibly
organs can be obtained from cloned fetuses as they develop through
organogenesis. Cells, tissues, and organs can be isolated from cloned
offspring as well. This process can provide a source of "materials" for
many medical and veterinary therapies including cell and gene therapy. If
the cells are transferred back into the animal in which the cells were
derived, then immunological rejection is averted. Aiso, because many cell
types can be isolated from these clones, other methodologies such as
2o hematopoietic chimerism can be used to avoid immunological rejection
among animals of the same species as well as between species.
Thus, in one aspect, the present invention provides a method for
cloning a mammal. In general, the mammal will be produced by a nuclear
transfer process comprising the following steps:
(i) obtaining desired non-serum starved, differentiated mammalian
cells to be used as a source of donor nuclei;
(ii) obtaining oocytes from a mammal of the same species as the cells
which are the source of donor nuclei;
(iii) enucieating said oocytes;
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(iv) transferring the desired differentiated cell or cell nucleus into the
enucieated oocyte, e.g., by fusion or injection, to form NT units;
(v) activating the resultant NT unit; and
(vi) transferring said cultured NT unit to a host mammal such that the
NT unit develops into a fetus.
Preferably, the activated nuclear transfer unit is cultured until greater
than the 2-cell developmental stage.
The present invention also includes a method of cloning a genetically
engineered or transgenic mammal, by which a desired DNA sequence is
16 inserted, removed or modified in the differentiated mammalian cell or cell
nucleus prior to insertion of the differentiated mammalian cell or cell
nucleus into the enucleated oocyte.
Also provided by the present invention are mammals obtained
according to the above method, and offspring of those mammals. The
present invention is preferably used for cloning ungulates.
The present invention further provides for the use of NT fetuses and
NT and chimeric offspring in the area of cell, tissue and organ
transplantation.
In another aspect, the present invention provides a method for
2 o producing CICM cells. The method comprises:
(i) inserting a desired non-serum starved, differentiated
mammalian cell or cell nucleus into an enucleated mammalian oocyte of the
same species as the differentiated cell or cell nucleus, under conditions
suitable for the formation of a nuclear transfer (NT) unit;
(ii) activating the resultant nuclear transfer unit; and
(iii) culturing cells obtained from said cultured NT unit to obtain CICM
cells.
Preferably, the activated nuclear transfer unit is cultured until greater
than the 2-cell developmental stage.
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The CICM cells are advantageously used in the area of cell, tissue
and organ transplantation, or in the production of fetuses or offspring,
including transgenic fetuses or offspring.
As used herein, a fetus is the unborn young of a viviporous animal
after it has taken form in the uterus. Thus, in cows the fetal stage occurs
from 35 days after conception until birth. In pigs, the fetal stage occurs
from 30 days after conception until birth. A mammal is an adult from birth
until death.
Preferably, the NT units will be cultured to a size of at least 2 to 400
1 a cells, preferably 4 to 128 cells, and most preferably to a size of at
least
about 50 cells.
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,
Theriogenoiogy, 43:181 (7995); Collas et al, Mol. ReportDev., 38:264-
267 (1994); Keefer et al, Biol. Reprod., 50:935-939 (1994); Sims et al,
Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993); WO 94/26884; WO
94/24274, and WO 90/03432, which are incorporated by reference in their
entirety herein. Also, U.S. Patent Nos. 4,944,384 and 5,057,420 describe
procedures for bovine nuclear transplantation.
Differentiated refers to cells having a different character or function
from the surrounding structures or from the cell of origin. Differentiated
mammalian cells are those cells which are past the early embryonic stage.
More particularly, the differentiated cells are those from at least past the
embryonic disc stage (day 10 of bovine embryogenesis). The differentiated
cells may be derived from ectoderm, mesoderm or endoderm.
Mammalian cells, including human cells, may be obtained by well
known methods. Mammalian cells useful in the present invention include,
by way of example, epithelial cells, neural cells, epidermal cells,
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keratinocytes, hematopoietic cells, melanocytes, chondrocytes,
lymphocytes (B and T lymphocytes), erythrocytes, macrophages,
monocytes, mononuclear cells, fibroblasts, cardiac muscle cells, and other
muscle cells, etc. Moreover, the mammalian cells used for nuclear transfer
may be obtained from different organs, e.g., skin, lung, pancreas, liver,
stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and
other urinary organs, etc. These are just examples of suitable donor cells.
Suitable donor cells, i.e., cells useful in the subject invention, may be
obtained from any cell or organ of the body. This includes all somatic or
to germ cells.
Fibroblast cells are an ideal cell type because they can be obtained
from developing fetuses and adult animals in large quantities. Fibroblast
cells are differentiated somewhat and, thus, were previously considered a
poor cell type to use in cloning procedures. Importantly, these cells can be
easily propagated in vitro with a rapid doubling time and can be clonally
propagated for use in gene targeting procedures. Again the present
invention is novel because differentiated cell types are used. The present
invention is advantageous because the cells can be easily propagated,
genetically modified and selected in vitro.
2o Other reported cloning methods (e.g., Wilmut et al, 1997) have relied
on the use of serum starved cells. In the present invention, however, the
donor cells are not in a state of serum starvation. According to Wilmut et
al (1997), serum starved cells are quiescent, i.e., exiting the growth phase.
Other methods (chemical, temperature, etc.) are also capable of producing
quiescent cells. The donor cells used in the present invention are not
quiescent.
Suitable mammalian sources for oocytes include sheep, cows, pigs,
goats, horses, rabbits, guinea pigs, mice, hamsters, rats, primates, etc.
Preferably, the oocytes will be obtained from ungulates, and most
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preferably bovine.
Methods for isolation of oocytes are well known in the art.
Essentially, this will comprise isolating oocytes from the ovaries or
reproductive tract of a mammal, 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.
1~ This process generally requires collecting immature (prophase I) oocytes
from mammalian ovaries, e.g., bovine ovaries obtained at a slaughterhouse,
and maturing the oocytes in a maturation medium prior to fertilization or
enucleation until the oocyte attains the metaphase II stage, which in the
case of bovine oocytes generally occurs about 18-24 hours post-aspiration.
For purposes of the present invention, this period of time is known as the
"maturation period." As used herein for calculation of time periods,
"aspiration" refers to aspiration of the immature oocyte from ovarian
follicles.
Additionally, metaphase II stage oocytes, which have been matured
2o in vivo have been successfully used in nuclear transfer techniques.
Essentially, mature metaphase II oocytes are collected surgically from
either non-superovulated or superovulated cows or heifers 35 to 48 hours
past the onset of estrus or past the injection of human chorionic
gonadotropin (hCG) or similar hormone.
The stage of maturation of the oocyte at enucleation and nuclear
transfer has been reported to be significant to the success of NT methods.
(See e.g., Prather et al., Differentiation, 48, 1-8, 1991). In general,
successful mammalian embryo cloning practices use the metaphase II
stage oocyte as the recipient oocyte because at this stage it is believed that
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the oocyte can be or is sufficiently "activated" to treat the introduced
nucleus as it does a fertilizing sperm. In domestic animals, and especially
cattle, the oocyte activation period generally ranges from about 16-52
hours, preferably about 28-42 hours post-aspiration.
For example, immature oocytes may be washed in HEPES buffered
hamster embryo culture medium (HECM) as described in Seshagine et al.,
Biol. Reprod., 40, 544-606, 1989, and then placed into drops of maturation
medium consisting of 50 micro(iters 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 ranges from about 10 to
40 hours, and preferably about 16-18 hours, the oocytes will be
enucleated. Prior to enucleation the oocytes will preferably be removed and
placed in HECM containing I milligram per milliliter of hyaluronidase prior to
removal of cumulus cells. This may be effected by repeated pipetting
through very fine bore pipettes or by vortexing briefly. The stripped
oocytes are then screened for polar bodies, and the selected metaphase II
oocytes, as determined by the presence of polar bodies, are then used for
2 o nuclear transfer. Enucleation follows.
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 an embryo
culture medium such as 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.
Enucleation may be accomplished microsurgically using a
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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 enuc(eated can then be
placed in a suitable culture medium, e.g., CR1aa plus 10% serum.
In the present invention, the recipient oocytes will preferably be
enucleated at a time ranging from about 10 hours to about 40 hours after
1-o the initiation of in vitro maturation, more preferably from about 16 hours
to
about 24 hours after initiation of in vitro maturation, and most preferably
about 16-18 hours after initiation of in vitro maturation.
A single mammalian cell of the same species as the enucleated
oocyte will then be transferred into the perivitelline space of the enucleated
oocyte used to produce the NT unit. The mammalian cell and the
enucleated oocyte will be used to produce NT units according to methods
known in the art. For example, the cells may be fused by electrofusion.
Electrofusion is accomplished by providing a pulse of electricity that is
sufficient to cause a transient breakdown of the plasma membrane. This
2 o breakdown of the plasma membrane is very short because the membrane
reforms rapidly. Thus, if two adjacent membranes are induced to
breakdown and upon reformation the lipid bilayers intermingle, small
channels will open between the two cells. Due to the thermodynamic
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.,
f 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 Sertdai virus as a fusogenic agent (Graham,
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blister lnot. Symp. Monogr., 9, 19, 19691.
Also, in some cases (e.g. with small donor nuclei) it may be
preferable to inject the nucleus directly into the oocyte rather than using
electroporation fusion. Such techniques are disclosed in Collas and Barnes,
Mol. Reprod. Dev., 38:264-267 (19941, incorporated by reference in its
entirety herein.
Preferably, the mammalian cell and oocyte are eiectrofused in a 500
,um chamber containing fusion medium (0.25 M D-sorbitoi, 100 ,uM calcium
acetate, 0.5 mM magnesium acetate, 1.0 g/I BSA (fatty acid free), G t-I 7.2)
10- by application of an electrical pulse of 90-120V for about 15 Nsec, about
24 hours after initiation of oocyte maturation. After fusion, the resultant
fused NT units are then placed in a suitable medium until activation, e.g.,
CR 1 as medium. Typically activation will be effected shortly thereafter,
typically less than 24 hours later, and preferably about 2-9 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
2 o conditions to which embryos are normally exposed.
Alternatively, activation may be achieved by application of known
activation agents. For example, penetration of oocytes by sperm during
fertilization has been shown to activate prefusion oocytes to yield greater
numbers of viable pregnancies and multiple genetically identical calves after
nuclear transfer. Also, treatments such as electrical and chemical shock
may 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., herein incorporated by reference in its entirety.
Additionally, activation may be effected by simultaneously or
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sequentially:
(i) increasing levels of divalent cations in the oocyte, and
(ii) reducing phosphoryiation of cellular proteins in the oocyte.
This will generally be effected by introducing divalent cations into the
oocyte cytoplasm, e.g., magnesium, strontium, barium or calcium, e.g., in
the form of an ionophore. Other methods of increasing divalent cation
levels include the use of electric shock, treatment with ethanol and
treatment with caged chelators.
Phosphoryiation may be reduced by known methods, e.g., by the
to addition of kinase inhibitors, e.g., serine-threonine kinase inhibitors,
such as
6-dimethylaminopurine, 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.
In one embodiment, NT activation is effected by briefly exposing the
fused NT unit to a TL-HEPES medium containing 5NM ionomycin and 1
mg/ml BSA, followed by washing in TL-HEPES containing 30 mg/ml BSA
within about 24 hours after fusion, and preferably about 2 to 9 hours after
fusion.
The activated NT units may then be cultured in a suitable in vitro
culture medium until the generation of CICM cells and cell colonies. Culture
media suitable for culturing and maturation of embryos are well known in
the art. Examples of known media, which may be used for bovine embryo
culture and maintenance, include Ham's F-10 + 10% fetal calf serum
(FCS), Tissue Culture Medium-199 (TCM-199) + 10% fetal calf serum,
Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's Phosphate Buffered
Saline (PBS), Eagle's and Whitten's media. One of the most common
media used for the collection and maturation of oocytes is TCM-199, and 1
to 20% serum supplement including fetal calf serum, newborn serum,
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estrual cow serum, Iamb serum or steer serum. A preferred maintenance
medium includes TCM-199 with Earl salts, 10% fetal calf serum, 0.2 mM
Na pyruvate and 50 ,ug/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.
Another maintenance medium is described in U.S. Patent 5,096,822
to Rosenkrans, Jr. et al., which is incorporated herein by reference. This
embryo medium, named CR1, contains the nutritional substances necessary
to support an embryo.
to CR1 contains hemicaicium L-lactate in amounts ranging from 1.0 mM
to 10 mM, preferably 1.0 mM to 5.0 mM. Hemicaicium L-lactate is L-
lactate with a hemicalcium salt incorporated thereon. Hemicalcium L-
lactate is significant in that a single component satisfies two major
requirements in the culture medium: (i) the calcium requirement necessary
for compaction and cytoskeleton arrangement; and (ii) the lactate
requirement necessary for metabolism and electron transport. Hemicalcium
L-lactate also serves as valuable mineral and energy source for the medium
necessary for viability of the embryos.
Advantageously, CR1 medium does not contain serum, such as fetal
2 o calf serum, and does not require the use of a co-culture of animal cells
or
other biological media, i.e., media comprising animal cells such as oviductal
cells. Biological media can sometimes be disadvantageous in that they may
contain microorganisms or trace factors which may be harmful to the
embryos and which are difficult to detect, characterize and eliminate.
Examples of the main components in CR1 medium include
hemicaicium L-lactate, sodium chloride, potassium chloride, sodium
bicarbonate and a minor amount of fatty-acid free bovine serum albumin
(Sigma A-6003). Additionally, a defined quantity of essential and non-
essential amino acids may be added to the medium. CR1 with amino acids
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is known by the abbreviation "CR1 aa."
CR1 medium preferably contains the following components in the
following quantities:
sodium chloride - 114.7 mM
potassium chloride - 3.1 mM
sodium bicarbonate - 26.2 mM
hemicalcium L-lactate - 5 mM
fatty-acid free BSA - 3 mg/ml
In one embodiment, the activated NT embryos unit are placed in
l0~ CR1 as medium containing 1 .9 mM DMAP for about 4 hours followed by a
wash in HECM and then cultured in CR1 as containing BSA.
For example, the activated NT units may be transferred to CR1 as
culture medium containing 2.0 mM DMAP (Sigma) and cultured under
ambient conditions, e.g., about 38.5'C, 5% COZ for a suitable time, e.g.,
about 4 to 5 hours.
Afterward, the cultured NT unit or units are preferably washed and
then placed in a suitable media, e.g., CR1 as medium containing 10% FCS
and 6 mg/ml contained in well plates which preferably contain a suitable
confluent feeder layer. Suitable feeder layers include, by way of example,
2o fibroblasts and epithelial cells, e.g., fibroblasts and uterine epithelial
cells
derived from ungulates, chicken fibroblasts, murine (e.g., mouse or rat)
fibrobiasts, STO and SI-m220 feeder cell lines, and BRL cells.
In one embodiment, the feeder cells comprise mouse embryonic
fibroblasts. Preparation of a suitable fibroblast feeder layer is 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 (5 x 105 ceils/ml) until
the NT units reach a size suitable far transferring to a recipient female, or
for obtaining cells which may be used to produce C1CM cells or cell
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colonies. Preferably, these NT units will be cultured until at least about 2
to 400 cells, more preferably about 4 to 128 cells, and most preferably at
least about 50 cells. The culturing will be effected under suitable
conditions, i.e., about 38.5 °C and 5 % CO2, with the culture medium
changed in order to optimize growth typically about every 2-5 days,
preferably about every 3 days.
The methods for embryo transfer and recipient animal management in
the present invention are standard procedures used in the embryo transfer
industry. Synchronous transfers are important for success of the present
invention, i.e., the stage of the NT embryo is in synchrony with the estrus
cycle of the recipient female. This advantage and how to maintain
recipients are reviewed in Siedel, G.E., Jr. ("Critical review of embryo
transfer procedures with cattle" in Fertilization and Embryonic Development
in Vitro (1981 ) L. Mastroianni, Jr, and J.D. Biggers, ed., Plenum Press,
New York, NY, page 3231, the contents of which are hereby incorporated
by reference.
By the present invention, cloning efficiency using nuclei donated from
adult cells may be the same as when using nuclei from fetal cells. For
example, the efficiency of development to morula and blastocyst stage
2 0 embryos is the same whether using nuclei from fetal or adult cow cells.
The present invention can also be used to clone genetically
engineered or transgenic mammals. As explained above, the present
invention is advantageous in that transgenic procedures can be simplified
by working with a differentiated cell source that can be clonally propagated.
In particular, the differentiated cells used for donor nuclei have a desired
DNA sequence inserted, removed or modified. Those genetically altered,
differentiated cells are then used for nuclear transplantation with enucleated
oocytes.
Any known method for inserting, deleting or modifying a desired
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DNA sequence from a mammalian cell may be used for altering the
differentiated cell to be used as the nuclear donor. These procedures may
remove all or part of a DNA sequence, and the DNA sequence may be
heterologous. Included is the technique of homologous recombination,
which allows the insertion, deletion or modification of a DNA sequence or
sequences at a specific site or sites in the cell genome.
The present invention can thus be used to provide adult mammals
with desired genotypes. Multiplication of adult ungulates with proven
genetic superiority or other desirable traits is particularly useful,
including
1 o transgenic or genetically engineered animals, and chimeric animals.
Furthermore, cells and tissues from the NT fetus, including transgenic
and/or chimeric fetuses, can be used in cell, tissue and organ
transplantation for the treatment of numerous diseases as described below
in connection with the use of CICM cells.
For production of CICM cells and cell lines, after NT units of the
desired size are obtained, the cells are mechanically removed from the zone
and are then used. 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.,
2 o irradiated fibroblast cells. Typically, the cells used to obtain the stem
cells
or cell colonies will be obtained from the inner most portion of the cultured
NT unit which is preferably at least 50 cells in size. However, NT units of
smaller or greater cell numbers as well as cells from other portions of the
NT unit may also be used to obtain ES cells and cell colonies. The cells are
2 5 maintained in the feeder layer in a suitable growth medium, e.g., alpha
MEM supplemented with 10% FCS and 0.1 mM f3-mercaptoethanol (Sigma)
and L-glutamine. The growth medium is changed as often as necessary to
optimize growth, e.g., about every 2-3 days.
This culturing process results in the formation of CICM cells or cell
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lines. One skilled in the art can vary the culturing conditions as desired to
optimize growth of the particular CICM cells. Also, genetically engineered
or transgenic mammalian CICM cells may be produced according to the
present invention. That is, the methods described above can be used to
produce NT units in which a desired DNA sequence or sequences have been
introduced, or from which all or part of an endogenous DNA sequence or
sequences have been removed or modified. Those genetically engineered or
transgenic NT units can then be used to produce genetically engineered or
transgenic CICM cells, including human cells.
The resultant ClCM cells and cell lines, preferably human CICM cells
and cell lines, have numerous therapeutic and diagnostic applications. Most
especially, such CICM cells may be used for cell transplantation therapies.
Human CICM cells have application in the treatment of numerous disease
conditions. Human NT units per se may also be used in the treatment of
disease conditions.
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 CICM cells produced according to the
invention should possess similar differentiation capacity. The CICM cells
2o according to the invention will be induced to differentiate to obtain the
desired cell types according to known methods. For example, the subject
human CICM cells may be induced to differentiate into hematopoietic stem
cells, nerve 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
CICM 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
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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
1 o muscle, nerve cells, among others.
Further, Bain et al, Dev. Biol., 168:342-357 (1995) teaches in vitro
differentiation of embryonic stem cells to produce neural cells which
possess neuronal properties. These references are exemplary of reported
methods for obtaining differentiated cells from embryonic or stem cells.
These references and in particular the disclosures therein relating to
methods for differentiating embryonic stem cells are incorporated by
reference in their entirety herein.
Thus, using known methods and culture medium, one skilled in the
art may culture the subject CICM cells, including genetically engineered or
2o transgenic CiCM cells, to obtain desired differentiated cell types, e.g.,
neural cells, muscle cells, hematopoietic cells, etc.
The subject CICM cells may be used to obtain any desired
differentiated cell type. Therapeutic usages of such differentiated human
cells are unparalleled. For example, human hematopoietic stem cells may
be used in medical treatments requiring bone marrow transplantation. Such
procedures are used to treat many diseases, e.g., late stage cancers such
as ovarian cancer and leukemia, as well as diseases that compromise the
immune system, such as AIDS. Hematopoietic stem cells can be obtained,
e.g., by fusing adult somatic cells of a cancer or AIDS patient, e.g.,
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epithelial cells or lymphocytes with an enucleated oocyte, obtaining CICM
cells as described above, and culturing such cells under conditions which
favor differentiation, until hematopoietic stem cells are obtained. Such
hematopoietic cells may be used in the treatment of diseases including
cancer and AIDS.
Alternatively, adult somatic cells from a patient with a neurological
disorder may be fused with an enucleated oocyte, human CICM cells
obtained therefrom, and such cells cultured under differentiation conditions
to produce neural cell lines. Specific diseases treatable by transplantation
of such human neural cells include, by way of example, Parkinson's
disease, Alzheimer's disease, ALS and cerebral palsy, among others. In the
specific case of Parkinson's disease, it has been demonstrated that
transplanted fetal brain neural cells make the proper connections with
surrounding cells and produce dopamine. This can result in long-term
reversal of Parkinson's disease symptoms.
The great advantage of the subject invention is that it provides an
essentially limitless supply of isogenic or syngenic human cells suitable for
transplantation. Therefore, it will obviate the significant problem associated
with current transplantation methods, i.e., rejection of the transplanted
2 o tissue which may occur because of host-vs-graft or graft-vs-host
rejection.
Conventionally, rejection is prevented or reduced by the administration of
anti-rejection drugs such as cyclosporine. 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.
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., hyperchofesterolemia,
heart diseases, cartilage replacement, burns, foot ulcers, gastrointestinal
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diseases, vascular diseases, kidney disease, urinary tract disease, and aging
related diseases and conditions.
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 DNA sequence encoding brain derived growth factor may be
introduced into human CICM 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 (myobiast and
fibroblast) derived cells. For example, astrocytes have been transfected
with BDNF gene using retroviral vectors, and the cells grafted into a rat
model of Parkinson's disease (Yoshimoto et ai., 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.
2o Neurol., 139:39-53 (1996) and references cited therein).
However, such ex vivo systems have problems. In particular,
retroviral vectors currently used are down-regulated in vivo and the
transgene is only transiently expressed (review by Mulligan, Science,
260:926-932 (1993)). Also, such studies used primary cells, astrocytes,
which have finite life span and replicate slowly. Such properties adversely
affect the rate of transfection and impede selection of stably transfected
cells. Moreover, it is almost impossible to propagate a large population of
gene targeted primary cells to be used in homologous recombination
techniques. By contrast, the difficulties associated with retroviral systems
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should be eliminated by the use of mammalian cloning using differentiated
cells and C1CM cells.
DNA sequences which may be introduced into the subject C1CM or
differentiated cells include, by way of example, those which encode
epidermal growth factor, basic fibrobiast growth factor, glial derived
neurotrophic growth factor, insulin-like growth factor (I and II),
neurotrophin-3, neurotrophin-4/5, ciliary neurotrophic factor, AFT-1,
cytokines (interleukins, interferons, colony stimulating factors, tumor
necrosis factors (alpha and beta), etc.), therapeutic enzymes, etc.
- in addition to the use of human NT units and CICM cells in cell,
tissue and organ transplantation, the present invention also includes the use
of non-human cells in the treatment of human diseases. Thus, CiCM cells,
NT fetuses and NT and chimeric offspring (transgenic or non-transgenic) of
any species may be used in the treatment of human disease conditions
where cell, tissue or organ transplantation is warranted. In general, CICM
cell, fetuses and offspring according to the present invention can be used
within the same species (autologous, syngenic or aliografts) or across
species (xenografts). For example, brain cells from bovine NT fetuses may
be used to treat Parkinson's disease.
2 o Also, the subject C1CM cells, preferably human cells, 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 CICM cells may be
used in drug studies.
Further, the subject CICM cells may be used as nuclear donors for
the production of other CICM cells and cell colonies.
In order to more clearly describe the subject invention, the following
examples are provided.
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EXAMPLE 1
Isolation of primary cultures of bovine and porcine embryonic and adult
bovine fibroblast cells.
Primary cultures of bovine and porcine fibroblasts were obtained from
fetuses (45 days of pregnancy for cattle and 35 days for pig fetuses). The
head, liver, heart and alimentary tract were aseptically removed, the fetuses
minced and incubated for 30 minutes at 37°C in prewarmed trypsin EDTA
solution (0.05% trypsin/0.02% EDTA; GIBCO, Grand Island, NY).
to Fibroblast cells were plated in tissue culture dishes and cultured in alpha-
MEM, medium (BioWhittaker, Walkersville, MD) supplemented with 10%
fetal calf serum (FCS) (Hyclone, Logen, UT), penicillin (100 IU/ml) and
streptomycin (50 ,ul/ml). The fibroblasts were grown and maintained in a
humidified atmosphere with 5% COZ in air at 37°C. Cells were passaged
regularly upon reaching confluency.
Adult fibroblast cells were isolated from the lung and skin of a cow
(approximately five years of age). Minced lung tissue was incubated
overnight at 10°C in trypsin EDTA solution (0.05% trypsin/0.02% EDTA;
GIBCO, Grand Island, NY). The following day tissue and any disassociated
2 o cells were incubated for one hour at 37 °C in prewarmed trypsin
EDTA
solution (0.05% trypsin/0.02% EDTA; GIBCO, Grand Island, NY) and
processed through three consecutive washes and trypsin incubations (one
hr). Fibroblast cells were plated in tissue culture dishes and cultured in
alpha-MEM medium (BioWhittaker, Walkersville, MD) supplemented with
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time in development, ranging from approximately post embryonic disc stage
through adult fife of the animal (bovine day 12 to 15 after fertilization to
10
to 15 years of age animals). This procedure can also be used to isolate
fibroblasts from other mammals, including mice.
Introduction of a marker gene (foreign heteroiogous DNA) into embryonic
and adult fibroblast cells.
The following electroporation procedure was conducted for both
embryonic (cattle and pigs) and adult (cattle) fibroblast cells. Standard
to microinjection procedures may also be used to introduce heterologous DNA
into fibroblast cells, however, in this example eiectroporation was used
because it is an easier procedure.
Culture plates containing propagating fibroblast cells were incubated
in trypsin EDTA solution (0.05% trypsin/0.02% EDTA; GIBCO, Grand
Island, NY) until the cells were in a single cell suspension. The cells were
spun down at 500 x g and re-suspended at 5 million cells per ml with
phosphate buffered saline (PBS).
The reporter gene construct contained the cytomegalovirus promoter
and the beta-gaiactosidase, neomycin phosphotransferase fusion gene
(beta-GEO). The reporter gene and the cells at 50 Ng/ml final concentration
were added to the electroporation chamber. After the electroporation
pulse, the fibroblast cells were transferred back into the growth medium
(alpha-MEM medium (BioWhittaker, Walkersville, MD) supplemented with
10% fetal calf serum (FCS) (Hyclone, l_ogen, UT), penicillin (100 IU/ml) and
streptomycin (50,u1/ml)).
The day after electroporation, attached fibroblast cells were selected
for stable integration of the reporter gene. 6418 (400 ,ug/ml) was added to
growth medium for 15 days (range: 3 days until the end of the cultured
cells' life span). This drug kills any cells without the beta-GEO gene, since
3 o they do not express the neo resistance gene. At the end of this time,
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colonies of stable transgenic cells were present. Each colony was
propagated independently of each other. Transgenic fibroblast cells were
stained with X-gal to observe expression of beta-galactosidase, and
confirmed positive for integration using PCR amplification of the beta-GEO
gene and run out on an agarose gel.
Use of transgenic fibroblast cells in nuclear transfer procedures to create
CICM cell lines and transgenic fetuses
lfl One line of cells (CL-1 ) derived from one colony of bovine embryonic
fibroblast cells was used as donor nuclei in the nuclear transfer (NT)
procedure. General NT procedures are described above.
Slaughterhouse oocytes were matured in vitro. The oocytes were
stripped of cumulus cells and enucleated with a beveled micropipette at
approximately 18 to 20 hrs post maturation (hpm). Enucleation was
confirmed in TL-HEPES medium plus Hoechst 33342 (3,ug/ml; Sigma).
Individual donor cells (fibrobiasts) were then placed in the perivitelline
space
of the recipient oocyte. The bovine oocyte cytoplasm and the donor
nucleus (NT unit) were fused together using electrofusion techniques. One
2 o fusion pulse consisting of 120 V for 15 Nsec in a 500 ,um gap chamber
filled with fusion medium was applied to the NT unit. This occurred at 24
hpm. The NT units were placed in CR1 as medium until 2fi to 27 hpm.
The general procedure used to artificially activate oocytes has been
described above. NT unit activation was initiated between 26 and 27 hpm.
Briefly, NT units were exposed for four min to ionomycin (5 ~M;
CaIBiochem, 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. Throughout the ionomycin treatment, NT units were also exposed to
2 mM DMAP (Sigma). Following the wash, NT units were then transferred
3o into a microdrop of CR1 as culture medium containing 2 mM DMAP (Sigma)
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and cultured at 38.5 ° C 5 % COZ for four to five hrs. The embryos were
washed and then placed in CR1 as medium plus 10% FCS and 6 mg/ml BSA
in four well plates containing a confluent feeder layer of mouse embryonic
fibroblast. The NT units were cultured for three more days at 38.5°C
and
5 % C02. Culture medium was changed every three days until days 5 to 8
after activation. At this time blastocyst stage NT embryos can be used to
produce transgenic CICM (cultured inner cell mass) cell lines or fetuses.
The inner cell mass of these NT units can be isolated and plated on a feeder
Layer. Also, NT units were transferred into recipient females. The
lo- pregnancies were aborted between 35-48 days of gestation. This resulted
in seven cloned transgenic fetuses having the beta-GEO gene in ail tissues
checked. Six of the seven embryos had a normal heart beat detected via
ultrasound observation. Also, histological sections of fetuses showed no
overt anomalies. Thus, this is a fast and easy method of making transgenic
CICM cell lines and fetuses. This procedure is generally conducive to gene
targeted CICM cell lines and fetuses.
The table below summarizes the results of these experiments.
CA 02294916 1999-12-29
WO 99/01163 PCT/US98/12800
-35-
a
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SUBSTITUTE SHEET (RULE 26~
CA 02294916 1999-12-29
WO 99/01163 PCT/US98/12800
-36-
EXAMPLE 2
Chimeric fetuses and offspring derived from transgenic CICM cells. The
transgenic CICM cell line was derived originally from a transgenic NT unit
(differentiated cell).
A CICM line derived from transgenic NT embryos (a CL-1 cell
transferred into an enucleated oocyte) was used to produce chimeric
embryos and fetuses. Colonies of transgenic CICM cells were
disaggregated either using 1-5 mg/ml pronase or 0.05 % trypsin/EDTA
combined with mechanical disaggregation methods so that clumps of five
-to or fewer cells were produced. Trypsin or pronase activity was inactivated
by passing the cells through multiple washes of 30 to 100% fetal calf
serum. The disaggregated cells were placed in micromanipulation plates
containing TL-HEPES medium. Fertilized embryos were also placed in these
plates and micromanipulation tools were used to produce the chimeric
embryos. Eight to ten transgenic CICM cells were injected into 8-16 cell
stage fertilized embryos. These embryos were cultured in vitro to the
blastocyst stage and then transferred into recipient animals.
A total of 6 blastocyst stage chimeric embryos were non-surgically
transferred into two recipient females. After five weeks of gestation 3
fetuses were recovered. Several tissues of the three fetuses, including
germ cells of the gonad (suggesting germ-line chimeras), were screened by
PCR amplification and southern blot hybridization of the amplified product
to a beta-galactosidase fragment. Of the three fetuses, two were positive
for contribution from the transgenic CICM cells. Both of these fetuses had
transgenic CICM contribution to the gonad.
Ten chimeric embryos were allowed to go to term, and 7 of the 10
developed to offspring. Ear notches were taken and DNA isolated from
each calf. Upon PCR amplification, one of the seven was confirmed to be a
transgenic chimeric offspring.
CA 02294916 1999-12-29
WO 99/01163 PCT/US98/12800
-37-
Transgenic NT embryos derived from transgenic CICM cell lines. The
transgenic CICM cell line was derived originally from a transgenic NT unit
(differentiated cell).
The same transgenic CICM cell lines were used to produce NT
embryos. The NT procedures described in Example 1 were used except
that CICM cells instead of fibroblast cells were used as the donor cell fused
with the enucleated oocyte. Colonies of transgenic CICM cells were
disaggregated either using 1-5 mg/ml pronase or 0.05 % trypsin/EDTA
combined with mechanical disaggregation methods so that clumps of five
or fewer cells were produced. Trypsin or pronase activity was inactivated
by passing the cells through multiple washes of 30 to 100% fetal calf
serum before transferring the cells into enucleated oocytes. Results are
reported in Table 1 (third group). Five blastocyst stage embryos were
produced.
