|Número de publicación||WO2001019977 A1|
|Tipo de publicación||Solicitud|
|Número de solicitud||PCT/US2000/025090|
|Fecha de publicación||22 Mar 2001|
|Fecha de presentación||14 Sep 2000|
|Fecha de prioridad||14 Sep 1999|
|También publicado como||CA2384413A1, CN1379814A, EP1214404A1, EP1214404A4, WO2001019977A9|
|Número de publicación||PCT/2000/25090, PCT/US/0/025090, PCT/US/0/25090, PCT/US/2000/025090, PCT/US/2000/25090, PCT/US0/025090, PCT/US0/25090, PCT/US0025090, PCT/US025090, PCT/US2000/025090, PCT/US2000/25090, PCT/US2000025090, PCT/US200025090, WO 0119977 A1, WO 0119977A1, WO 2001/019977 A1, WO 2001019977 A1, WO 2001019977A1, WO-A1-0119977, WO-A1-2001019977, WO0119977 A1, WO0119977A1, WO2001/019977A1, WO2001019977 A1, WO2001019977A1|
|Inventores||James Robl, Jose Cibelli, Steven L. Stice|
|Solicitante||University Of Massachusetts, A Public Institution Of Higher Education By The Commonwealth Of Massachusetts, As Represented By Its Amherst Campus|
|Exportar cita||BiBTeX, EndNote, RefMan|
|Citas de patentes (3), Otras citas (6), Citada por (8), Clasificaciones (34), Eventos legales (20)|
|Enlaces externos: Patentscope, Espacenet|
EMBRYONIC OR STEM-LIKE CELL LINES PRODUCED BY
CROSS SPECIES NUCLEAR TRANSPLANTATION AND
METHODS FOR ENHANCING EMBRYONIC DEVELOPMENT
BY GENETIC ALTERATION OF DONOR CELLS OR
BY TISSUE CULTURE CONDITIONS
CROSS-REFERENCE TO RELATES APPLICATIONS
This application claims priority under 35 U.S.C. §119 to PCT/US99/04608, filed
on March 2, 1999. Also, this application is a continuation-in-part of U.S. Serial No.
09/032,995, filed March 2, 1998, which is in turn a continuation-in-part of U.S. Serial No.
08/699,040, filed on August 19, 1996. All of these applications are incorporated by
reference in their entirety herein.
FIELD OF THE INVENTION
The present invention generally relates to the production of embryonic or stem-like
cells by the transplantation of cell nuclei derived from animal or human cells into
enucleated animal oocytes of a species different from the donor nuclei. The present
invention more specifically relates to the production of primate or human embryonic or
stem-like cells by transplantation of the nucleus of a primate or human cell into an
enucleated animal oocyte, e.g., a primate or ungulate oocyte and in a preferred
embodiment a bovine enucleated oocyte.
The present invention further relates to the use of the resultant embryonic or stem¬
like cells, preferably primate or human embryonic or stem-like cells for therapy, for diag-
nostic applications, for the production of differentiated cells which may also be used for therapy or diagnosis, and for the production of transgenic embryonic or transgenic
differentiated cells, cell lines, tissues and organs. Also, the embryonic or stem-like cells
obtained according to the present invention may themselves be used as nuclear donors in
nuclear transplantation or nuclear transfer methods for the production of chimeras or
clones, preferably transgenic cloned or chimeric animals.
BACKGROUND OF THE INVENTION
Methods for deriving embryonic stem (ES) cell lines in vitro from early
preimplantation mouse embryos are well known. (See, e.g., Evans et al., Nature, 29: 154-
156 (1981); Martin, Proc. Natl. Acad. Sci., USA, 78:7634-7638 (1981)). ES cells can be
passaged in an undifferentiated state, provided that a feeder layer of fibroblast cells
(Evans et al., Id.) or a differentiation inhibiting source (Smith et al., Dev. Biol, 121:1-9
(1987)) is present.
ES cells have been previously reported to possess numerous applications. For
example, it has been reported that ES cells can be used as an in vitro model for differen-
tiation, especially for the study of genes which are involved in the regulation of early
development. Mouse ES cells can give rise to germline chimeras when introduced into
preimplantation mouse embryos, thus demonstrating their pluripotency (Bradley et al.,
Nature, 309:255-256 (1984)).
In view of their ability to transfer their genome to the next generation, ES cells
have potential utility for germline manipulation of livestock animals by using ES cells
with or without a desired genetic modification. Moreover, in the case of livestock animals, e.g., ungulates, nuclei from like preimplantation livestock embryos support the
development of enucleated oocytes to term (Smith et al., Biol. Reprod., 40:1027-1035
(1989); and Keefer et al., Biol. Reprod., 50:935-939 (1994)). This is in contrast to nuclei
from mouse embryos which beyond the eight-cell stage after transfer reportedly do not
support the development of enucleated oocytes (Cheong et al, Biol. Reprod., 48:958
(1993)). Therefore, ES cells from livestock animals are highly desirable because they
may provide a potential source of totipotent donor nuclei, genetically manipulated or
otherwise, for nuclear transfer procedures.
Some research groups have reported the isolation of purportedly pluripotent
embryonic cell lines. For example, Notarianni et al., J. Reprod. Fert. Suppl, 43:255-260
(1991), report the establishment of purportedly stable, pluripotent cell lines from pig and
sheep blastocysts which exhibit some morphological and growth characteristics similar
to that of cells in primary cultures of inner cell masses isolated immunosurgically from
sheep blastocysts. (Id.) Also, Notarianni et al., J. Reprod. Fert. Suppl., 41:51-56 (1990)
discloses maintenance and differentiation in culture of putative pluripotential embryonic
cell lines from pig blastocysts. Further, Gerfen et al., Anim. Biotech, 6(1):1-14 (1995)
disclose the isolation of embryonic cell lines from porcine blastocysts. These cells are
stably maintained in mouse embryonic fibroblast feeder layers without the use of
conditioned medium. These cells reportedly differentiate into several different cell types
during culture (Gerfen et al., Id). Further, Saito et al., Roux's Arch. Dev. Biol, 201 : 134-141 (1992) report bovine
embryonic stem cell-like cell lines cultured which survived passages for three, but were
lost after the fourth passage. Still further, Handyside et al., Roux's Arch. Dev. Biol.,
196:185-190 (1987) disclose culturing of immunosurgically isolated inner cell masses of
sheep embryos under conditions which allow for the isolation of mouse ES cell lines
derived from mouse ICMs. Handyside et al. (1987) (Id.), report that under such condi¬
tions, the sheep ICMs attach, spread, and develop areas of both ES cell-like and
endoderm-like cells, but that after prolonged culture only endoderm-like cells are evident.
Recently, Cherny et al., Theriogenology, 41 :175 (1994) reported purportedly
pluripotent bovine primordial germ cell-derived cell lines maintained in long-term
culture. These cells, after approximately seven days in culture, produced ES-like colonies
which stain positive for alkaline phosphatase (AP), exhibited the ability to form embryoid
bodies, and spontaneously differentiated into at least two different cell types. These cells
also reportedly expressed mRNA for the transcription factors OCT4, OCT6 and HES1,
a pattern of homeobox genes which is believed to be expressed by ES cells exclusively.
Also recently, Campbell et al., Nature, 380:64-68 (1996) reported the production
of live lambs following nuclear transfer of cultured embryonic disc (ED) cells from day
nine ovine embryos cultured under conditions which promote the isolation of ES cell lines
in the mouse. The authors concluded based on their results that ED cells from day nine ovine embryos are totipotent by nuclear transfer and that totipotency is maintained in
Van Stekelenburg-Hamers et al., Mol. Reprod. Dev., 40:444-454 (1995), reported
the isolation and characterization of purportedly permanent cell lines from inner cell mass
cells of bovine blastocysts. The authors isolated and cultured ICMs from 8 or 9 day
bovine blastocysts under different conditions to determine which feeder cells and culture
media are most efficient in supporting the attachment and outgrowth of bovine ICM cells.
They concluded based on their results that the attachment and outgrowth of cultured ICM
cells is enhanced by the use of STO (mouse fibroblast) feeder cells (instead of bovine
uterus epithelial cells) and by the use of charcoal-stripped serum (rather than normal se¬
rum) to supplement the culture medium. Van Stekelenburg et al reported, however, that
their cell lines resembled epithelial cells more than pluripotent ICM cells. (Id.)
Still further, Smith et al., WO 94/24274, published October 27, 1994, Evans et al,
WO 90/03432, published April 5, 1990, and Wheeler et al, WO 94/26889, published
November 24, 1994, report the isolation, selection and propagation of animal stem cells
which purportedly may be used to obtain transgenic animals. Also, Evans et al.,
WO 90/03432, published on April 5, 1990, reported the derivation of purportedly
pluripotent embryonic stem cells derived from porcine and bovine species which
assertedly are useful for the production of transgenic animals. Further, Wheeler et al,
WO 94/26884, published November 24, 1994, disclosed embryonic stem cells which are
assertedly useful for the manufacture of chimeric and transgenic ungulates. Thus, based on the foregoing, it is evident that many groups have attempted to produce ES cell lines,
e.g., because of their potential application in the production of cloned or transgenic
embryos and in nuclear transplantation.
The use of ungulate ICM cells for nuclear transplantation has also been reported.
For example, Collas et al., Mol. Reprod. Dev., 38:264-267 (1994) disclose nuclear trans¬
plantation of bovine ICMs by microi jection of the lysed donor cells into enucleated
mature oocytes. The reference disclosed culturing of embryos in vitro for seven days to
produce fifteen blastocysts which, upon transferral into bovine recipients, resulted in four
pregnancies and two births. Also, Keefer et al., Biol. Reprod., 50:935-939 (1994),
disclose the use of bovine ICM cells as donor nuclei in nuclear transfer procedures, to
produce blastocysts which, upon transplantation into bovine recipients, resulted in several
live offspring. Further, Sims et al., Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993),
disclosed the production of calves by transfer of nuclei from short-term in vitro cultured
bovine ICM cells into enucleated mature oocytes.
Also, the production of live lambs following nuclear transfer of cultured
embryonic disc cells has been reported (Campbell et al., Nature, 380:64-68 (1996)). Still
further, the use of bovine pluripotent embryonic cells in nuclear transfer and the
production of chimeric fetuses has also been reported (Stice et al., Biol. Reprod., 54:100-
110 (1996)); Collas et al, Mol. Reprod. Dev., 38:264-267 (1994).
Further, there have been previous attempts to produce cross species NT units
(Wolfe et al., Theriogenology, 33:350 (1990). Specifically, bovine embryonic cells were fused with bison oocytes to produce some cross species NT units possibly having an inner
cell mass. However, embryonic cells, not adult cells were used, as donor nuclei in the
nuclear transfer procedure. The dogma has been that embryonic cells are more easily
reprogrammed than adult cells. This dates back to earlier NT studies in the frog (review
by DiBerardino, Differentiation, 17:17-30 (1980)). Also, this study involved very
phylogenetically similar animals (cattle nuclei and bison oocytes). By contrast, previ¬
ously when more diverse species were fused during NT (cattle nuclei into hamster
oocytes), no inner cell mass structures were obtained. Further, it has never been
previously reported that the inner cell mass cells from NT units could be used to form an
ES cell-like colony that could be propagated.
Also, Collas et al (Id.), taught the use of granulosa cells (adult somatic cells) to
produce bovine nuclear transfer embryos. However, unlike the present invention, these
experiments did not involve cross-species nuclear transfer. Also, unlike the present
invention ES-like cell colonies were not obtained.
Recently, U.S. Patent No. 5,843,780, issued to James A. Thomson on December
1, 1998, assigned to the Wisconsin Alumni Research Foundation, purports to disclose a
purified preparation of primate embryonic stem cells that are (i) capable of proliferation
in an in vitro culture for over one year; (ii) maintain a karyotype in which all
chromosomes characteristic of the primate species are present and not noticeably altered
through prolonged culture; (iii) maintains the potential to differentiate into derivatives of
endoderm, mesoderm and ectoderm tissues throughout culture; and (iv) will not differentiate when cultured on a fibroblast feeder layer. These cells were reportedly
negative for the SSEA-1 marker, positive for the SEA-3 marker, positive for the SSEA-4
marker, express alkaline phosphatase activity, are pluripotent, and have karyotypes which
include the presence of all the chromosomes characteristic of the primate species and in
which none of the chromosomes are altered. Further, these cells are respectfully positive
for the TRA-1-60, and TRA-1-81 markers. The cells purportedly differentiate into
endoderm, mesoderm and ectoderm cells when injected into a SCID mouse. Also,
purported embryonic stem cell lines derived from human or primate blastocysts are
discussed in Thomson et al., Science 282:1145-1147 and Proc. Natl. Acad. Sci., USA
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
In this regard, numerous human diseases have been identified which may be
treated by cell transplantation. For example, Parkinson's disease is caused by degenera¬
tion of dopaminergic neurons in the substantia nigra. Standard treatment for Parkinson's
involves administration of L-DOPA, which temporarily ameliorates the loss of dopamine,
but causes severe side effects and ultimately does not reverse the progress of the disease.
A different approach to treating Parkinson's, which promises to have broad applicability to treatment of many brain diseases and central nervous system injury, involves
transplantation of cells or tissues from fetal or neonatal animals into the adult brain. Fetal
neurons from a variety of brain regions can be incorporated into the adult brain. Such
grafts have been shown to alleviate experimentally induced behavioral deficits, including
complex cognitive functions, in laboratory animals. Initial test results from human
clinical trials have also been promising. However, supplies of human fetal cells or tissue
obtained from miscarriages is very limited. Moreover, obtaining cells or tissues from
aborted fetuses is highly controversial.
There is currently no available procedure for producing "fetal-like" cells from the
patient. Further, allograft tissue is not readily available and both allograft and xenograft
tissue are subject to graft rejection. Moreover, in some cases, it would be beneficial to
make genetic modifications in cells or tissues before transplantation. However, many
cells or tissues wherein such modification would be desirable do not divide well in culture
and most types of genetic transformation require rapidly dividing cells.
There is therefore a clear need in the art for a supply of human embryonic or stem¬
like undifferentiated cells for use in transplants and cell and gene therapies.
OBJECTS OF THE INVENTION
It is an object of the invention to provide novel and improved methods for
producing embryonic or stem-like cells. It is a more specific object of the invention to provide a novel method for
producing embryonic or stem-like cells which involves transplantation of the nucleus of
a mammalian or human cell into an enucleated oocyte of a different species.
It is another specific object of the invention to provide a novel method for
producing non-human primate or human embryonic or stem-like cells which involves
transplantation of the nucleus of a non-human primate or human cell into an enucleated
animal or human oocyte, e.g., an ungulate, human or primate enucleated oocyte.
It is another object of the invention to enhance the efficacy of cross-species nuclear
transfer by incorporating mitochondrial DNA derived from the same species (preferably
same donor) as the donor cell into the oocyte of a different species that is used for nuclear
transfer, before or after enucleation, or into the nuclear transfer unit (after the donor cell
has been introduced).
It is still another object of the invention to enhance the efficacy of cross-species
nuclear transfer by fusing an enucleated somatic cell (e.g., an enucleated human somatic
cell) (karyoplast) with an activated or non-activated, enucleated or non-enucleated oocyte
of a different species, e.g., bovine, or by fusion with an activated or unactivated cross-
species NT unit which may be cleaved or uncleaved.
It is another object of the invention to provide a novel method for producing
lineage-defective non-human primate or human embryonic or stem-like cells which
involves transplantation of the nucleus of a non-human primate or human cell, e.g., a
human adult cell into an enucleated non-human primate or human oocyte, wherein such cell has been genetically engineered to be incapable of differentiation into a specific cell
lineage or has been modified such that the cells are "mortal", and thereby do not give rise
to a viable offspring, e.g., by engineering expression of anti-sense or ribozyme telomerase
It is still another object of the invention to enhance efficiency of nuclear transfer
and specifically to enhance the development of preimplantation embryos produced by
nuclear transfer by genetically engineering donor somatic cells used for nuclear transfer
to provide for the expression of genes that enhance embryonic development, e.g., genes
of the MHC I family, and in particular Ped genes such as Q7 and/or Q9.
It is another object of the invention to enhance the production of nuclear transfer
embryos, e.g., cross-species nuclear transfer embryos, by the introduction of transgenes
before or after nuclear transfer that provide for the expression of an antisense DNA
encoding a cell death gene such as BAX, Apaf-1, or capsase, or a portion thereof, or
It is yet another object of the invention to enhance the production of nuclear
transfer embryos by TP and more specifically nuclear transfer embryos by genetically
altering the donor cell used for nuclear transfer such that it is resistant to apoptosis, e.g.,
by introduction of a DNA construct that provides for the expression of genes that inhibit
apoptosis, e.g., Bcl-2 or Bcl-2 family members and/or by the expression of antisense
ribozymes specific to genes that induce apoptosis during early embryonic development. It is still another object of the invention to improve the efficacy of nuclear transfer
by improved selection of donor cells of a specific cell cycle stage, e.g., Gl phase, by
genetically engineering donor cells such that they express a DNA construct encoding a
particular cyclin linked to a detectable marker, e.g., one that encodes a visualizable (e.g.,
fluorescent tag) marker protein.
It is also an object of the invention to enhance the development of in vitro
produced embryos, by culturing such embryos in the presence of one or more protease
inhibitors, preferably one or more capsase inhibitors, thereby inhibiting apoptosis.
It is another object of the invention to provide embryonic or stem-like cells
produced by transplantation of nucleus of an animal or human cell into an enucleated
oocyte of a different species.
It is a more specific object of the invention to provide primate or human
embryonic or stem-like cells produced by transplantation of the nucleus of a primate or
human cell into an enucleated animal oocyte, e.g., a human, primate or ungulate enucle-
It is another object of the invention to use such embryonic or stem-like cells for
therapy or diagnosis.
It is a specific object of the invention to use such primate or human embryonic or
stem-like cells for treatment or diagnosis of any disease wherein cell, tissue or organ
transplantation is therapeutically or diagnostically beneficial. It is another specific object of the invention to use the embryonic or stem-like cells
produced according to the invention for the production of differentiated cells, tissues or
It is a more specific object of the invention to use the primate or human embryonic
or stem-like cells produced accordmg to the invention for the production of differentiated
human cells, tissues or organs.
It is another specific object of the invention to use the embryonic or stem-like cells
produced according to the invention for the production of genetically engineered embry¬
onic or stem-like cells, which cells may be used to produce genetically engineered or
transgenic differentiated human cells, tissues or organs, e.g., having use in gene therapies.
It is another specific object of the invention to use the embryonic or stem-like cells
produced according to the invention in vitro, e.g. for study of cell differentiation and for
assay purposes, e.g. for drug studies.
It is another object of the invention to provide improved methods of
transplantation therapy, comprising the usage of isogenic or synegenic cells, tissues or
organs produced from the embryonic or stem-like cells produced according to the
invention. Such therapies include by way of example treatment of diseases and injuries
including Parkinson's, Huntington's, Alzheimer's, ALS, spinal cord injuries, multiple
sclerosis, muscular dystrophy, diabetes, liver diseases, heart disease, cartilage replace-
ment, burns, vascular diseases, urinary tract diseases, as well as for the treatment of
immune defects, bone marrow transplantation, cancer, among other diseases. It is another object of the invention to use the transgenic or genetically engineered
embryonic or stem-like cells produced according to the invention for gene therapy, in
particular for the treatment and/or prevention of the diseases and injuries identified,
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
It is still another object of the invention to use genetically engineered ES cells
produced according to the invention for the production of transgenic animals, e.g., non-
human primates, rodents, ungulates, etc. Such transgenic animals can be used to produce,
e.g., animal models for study of human diseases, or for the production of desired
polypeptides, e.g., therapeutics or nutripharmaceuticals.
With the foregoing and other objects, advantages and features of the invention that
will become hereinafter apparent, the nature of the invention may be more clearly
understood by reference to the following detailed description of the preferred
embodiments of the invention and to the appended claims.
BRIEFS DESCRIPTION OF THE FIGURES
Figure 1 is a photograph of a nuclear transfer (NT) unit produced by transfer of an
adult human cell into an enucleated bovine oocyte. Figures 2 to 5 are photographs of embryonic stem-like cells derived from a NT
unit such as is depicted in Figure 1.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a novel method for producing embryonic or stem-
like cells, and more specifically non-human primate or human embryonic or stem-like
cells by nuclear transfer or nuclear transplantation. In the subject application, nuclear
transfer or nuclear transplantation or NT are used interchangeably.
As discussed supra, the isolation of actual embryonic or stem-like cells by nuclear
transfer or nuclear transplantation has never been reported. Rather, previous reported
isolation of ES-like cells has been from fertilized embryos. Also, successful nuclear
transfer involving cells or DNA of genetically dissimilar species, or more specifically
adult cells or DNA of one species (e.g., human) and oocytes of another non-related
species has never been reported. Rather, while embryos produced by fusion of cells of
closely related species, has been reported, e.g., bovine-goat and bovine-bison, they did
not produce ES cells. (Wolfe et al, Theriogenology, 33(1 ):350 (1990).) Also, there has
never been reported a method for producing primate or human ES cells derived from a
non-fetal tissue source. Rather, the limited human fetal cells and tissues which are
currently available must be obtained or derived from spontaneous abortion tissues and
from aborted fetuses.
Also, prior to the present invention, no one obtained embryonic or stem-like cells
by cross-species nuclear transplantation. Quite unexpectedly, the present inventors discovered that human embryonic or
stem-like cells and cell colonies may be obtained by transplantation of the nucleus of a
human cell, e.g., an adult differentiated human cell, into an enucleated animal oocyte,
which is used to produce nuclear transfer (NT) units, the cells of which upon culturing
give rise to human embryonic or stem-like cells and cell colonies. This result is highly
surprising because it is the first demonstration of effective cross-species nuclear
transplantation involving the introduction of a differentiated donor cell or nucleus into an
enucleated oocyte of a genetically dissimilar species, e.g., the transplantation of cell
nuclei from a differentiated animal or human cell, e.g., adult cell, into the enucleated egg
of a different animal species, to produce nuclear transfer units containing cells which
when cultured under appropriate conditions give rise to embryonic or stem-like cells and
Preferably, the NT units used to produce ES-like cells will be cultured to a size of
at least 2 to 400 cells, preferably 4 to 128 cells, and most preferably to a size of at least
about 50 cells.
In the present invention, embryonic or stem-like cells refer to cells produced
according to the present invention. The present application refers to such cells as stem¬
like cells rather than stem cells because of the manner in which they are typically
produced, i.e., by cross-species nuclear transfer. While these cells are expected to possess
similar differentiation capacity as normal stem cells they may possess some insignificant
differences because of the manner they are produced. For example, these stem- like cells may possess the mitochondria of the oocytes used for nuclear transfer, and thus not
behave identically to conventional embryonic stem cells.
The present discovery was made based on the observation that nuclear
transplantation of the nucleus of an adult human cell, specifically a human epithelial cell
obtained from the oral cavity of a human donor, when transferred into an enucleated
bovine oocyte, resulted in the formation of nuclear transfer units, the cells of which upon
culturing gave rise to human stem-like or embryonic cells and human embryonic or stem¬
like cell colonies. This result has recently been reproduced by transplantation of
keratinocytes from an adult human into an enucleated bovine oocyte with the successful
production of a blastocyst and ES cell line. Based thereon, it is hypothesized by the
present inventors that bovine oocytes and human oocytes, and likely mammals in general
must undergo maturation processes during embryonic development which are sufficiently
similar or conserved so as to permit the bovine oocyte to function as an effective sub¬
stitute or surrogate for a human oocyte. Apparently, oocytes in general comprise factors,
likely proteinaceous or nucleic acid in nature, that induce embryonic development under
appropriate conditions, and these functions that are the same or very similar in different
species. These factors may comprise material RNAs and/or telomerase.
Based on the fact that human cell nuclei can be effectively transplanted into bovine
oocytes, it is reasonable to expect that human cells may be transplanted into oocytes of
other non-related species, e.g., other ungulates as well as other animals. In particular,
other ungulate oocytes should be suitable, e.g. pigs, sheep, horses, goats, etc. Also, oocytes from other sources should be suitable, e.g. oocytes derived from other primates,
amphibians, rodents, rabbits, guinea pigs, etc. Further, using similar methods, it should
be possible to transfer human cells or cell nuclei into human oocytes and use the resultant
blastocysts to produce human ES cells.
Therefore, in its broadest embodiment, the present invention involves the
transplantation of an animal or human cell nucleus or animal or human cell into an oocyte
(preferably enucleated) of an animal species different from the donor nuclei, by injection
or fusion, to produce an NT unit containing cells which may be used to obtain embryonic
or stem-like cells and/or cell cultures. Enucleation (removal of endogenous oocyte
nucleus) may be effected before or after nuclear transfer. For example, the invention may
involve the transplantation of an ungulate cell nucleus or ungulate cell into an enucleated
oocyte of another species, e.g., another ungulate or non-ungulate, by injection or fusion,
which cells and/or nuclei are combined to produce NT units and which are cultured under
conditions suitable to obtain multicellular NT units, preferably comprising at least about
2 to 400 cells, more preferably 4 to 128 cells, and most preferably at least about 50 cells.
The cells of such NT units may be used to produce embryonic or stem-like cells or cell
colonies upon culturing.
However, the preferred embodiment of the invention comprises the production of
non-human primate or human embryonic or stem-like cells by transplantation of the
nucleus of a donor human cell or a human cell into an enucleated human, primate, or non- primate animal oocyte, e.g., an ungulate oocyte, and in a preferred embodiment a bovine
In general, the embryonic or stem-like cells will be produced by a nuclear transfer
process comprising the following steps:
(i) obtaining desired human or animal cells to be used as a source of donor nuclei
(which may be genetically altered);
(ii) obtaining oocytes from a suitable source, e.g. a mammal and most preferably
a primate or an ungulate source, e.g. bovine,
(iii) enucleating said oocytes by removal of endogenous nucleus;
(iv) transferring the human or animal cell or nucleus into the enucleated oocyte
of an animal species different than the donor cell or nuclei, e.g., by fusion or injection,
wherein steps (iii) and (iv) may be effected in either order;
(v) culturing the resultant NT product or NT unit to produce multiple cell
structures (embryoid structures having a discernible inner cell mass); and
(vi) culturing cells obtained from said embryos to obtain embryonic or stem- like
cells and stem-like cell colonies.
Nuclear transfer techniques or nuclear transplantation techniques are known in the
literature and are described in many of the references cited in the Background of the
Invention. See, in particular, Campbell et al, Theriogenology, 43:181 (1995); Collas et
al, Mol. Report Dev., 38:264-267 (1994); Keefer et al, Biol. Reprod., 50:935-939 (1994);
Sims et al, Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993); WO 94/26884; WO 94/24274, and WO 90/03432, which are incorporated by reference in their entirety herein.
Also, U.S. Patent Nos. 4,944,384 and 5,057,420 describe procedures for bovine nuclear
transplantation. See, also Cibelli et al, Science, Vol. 280: 1256-1258 (1998).
Human or animal cells, preferably mammalian cells, may be obtained and cultured
by well known methods. Human and animal cells useful in the present invention include,
by way of example, epithelial, neural cells, epidermal cells, keratinocytes, hematopoietic
cells, melanocytes, chondrocytes, lymphocytes (B and T lymphocytes), other immune
cells, erythrocytes, macrophages, melanocytes, monocytes, mononuclear cells,
fibroblasts, cardiac muscle cells, and other muscle cells, etc. Moreover, the human cells
used for nuclear transfer may be obtained from different organs, e.g., skin, lung, pancreas,
liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra and other
urinary organs, etc. These are just examples of suitable donor cells. Suitable donor cells,
i.e., cells useful in the subject invention, may be obtained from any cell or organ of the
body. This includes all somatic or germ cells. Preferably, the donor cells or nucleus
would comprise actively dividing, i.e., non-quiescent, cells as this has been reported to
enhance cloning efficacy. Also preferably, such donor cells will be in the Gl cell cycle.
The resultant blastocysts may be used to obtain embryonic stem cell lines
according to the culturing methods reported by Thomson et al., Science, 282:1145-1147
(1998) and Thomson et al., Proc. Natl. Acad. Sci., USA, 92:7544-7848 (1995),
incorporated by reference in their entirety herein. In the Example which follows, the cells used as donors for nuclear transfer were
epithelial cells derived from the oral cavity of a human donor and adult human
keratinocytes. However, as discussed, the disclosed method is applicable to other human
cells or nuclei. Moreover, the cell nuclei may be obtained from both human somatic and
It is also possible to arrest donor cells at mitosis before nuclear transfer, using a
suitable technique known in the art. Methods for stopping the cell cycle at various stages
have been thoroughly reviewed in U.S. Patent 5,262,409, which is herein incorporated
by reference. In particular, while cycloheximide has been reported to have an inhibitory
effect on mitosis (Bowen and Wilson (1955) J. Heredity, 45:3-9), it may also be
employed for improved activation of mature bovine follicular oocytes when combined
with electric pulse treatment (Yang et al. (1992) Biol. Reprod., 42 (Suppl. 1): 117).
Zygote gene activation is associated with hyperacetylation of Histone H4.
Trichostatin-A has been shown to inhibit histone deacetylase in a reversible manner
(Adenot et al. Differential H4 acetylation of paternal and maternal chromatin precedes
DNA replication and differential transcriptional activity in pronuclei of 1-cell mouse
embryos. Development (Nov. 1997) 124(22): 4615-4625; Yoshida et al. Trichostatm A
and trapoxin: novel chemical probes for the role of histone acetylation in chromatin
structure and function. Bioessays (May, 1995) 17(5): 423-430), as have other compounds.
For instance, butyrate is also believed to cause hyper-acetylations of histones by
inhibiting histone deacetylase. Generally, butyrate appears to modify gene expression and in almost all cases its addition to cells in culture appears to arrest cell growth. Use of
butyrate in this regard is described in U.S. Patent No. 5,681,718, which is herein
incorporated by reference. Thus, donor cells may be exposed to Trichostatin-A or another
appropriate deacetylase inhibitor prior to fusion, or such a compound may be added to the
culture media prior to genome activation.
Additionally, demethylation of DNA is thought to be a requirement for proper
access of transcription factors to DNA regulatory sequences. Global demethylation of
DNA from the eight-cell stage to the blastocyst stage in preimplantation embryos has
previously been described (Stein et al., Mol. Reprod. & Dev., 47(4): 421-429). Also,
Jaenisch et al. (1997) have reported that 5-azacytidine can be used to reduce the level of
DNA methylation in cells, potentially leading to increased access of transcription factors
to DNA regulatory sequences. Accordingly, donor cells may be exposed to 5-azacytidine
(5-Aza) previous to fusion, or 5-Aza may be added to the culture medium from the 8 cell
stage to blastocyst. Alternatively, other known methods for effecting DNA demethylation
may be used.
The oocytes used for nuclear transfer may be obtained from animals including
mammals and amphibians. Suitable mammalian sources for oocytes include sheep,
bovines, ovines, pigs, horses, rabbits, goats, guinea pigs, mice, hamsters, rats, primates,
humans, etc. In the preferred embodiments, the oocytes will be obtained from primates
or ungulates, e.g., a bovine. Methods for isolation of oocytes are well known in the art. Essentially, this will
comprise isolating oocytes from the ovaries or reproductive tract of a mammal or
amphibian, e.g., a bovine. A readily available source of bovine oocytes is slaughterhouse
For the successful use of techniques such as genetic engineering, nuclear transfer
and cloning, oocytes must generally be matured in vitro before these cells may be used
as recipient cells for nuclear transfer, and before they can be fertilized by the sperm cell
to develop into an embryo. This process generally requires collecting immature
(prophase I) oocytes from animal ovaries, e.g., bovine ovaries obtained at a
slaughterhouse and maturing the oocytes in a maturation medium prior to fertilization or
enucleation until the oocyte attains the metaphase II stage, which in the case of bovine
oocytes generally occurs about 18-24 hours post-aspiration. For purposes of the present
invention, this period of time is known as the "maturation period." As used herein for
calculation of time periods, "aspiration" refers to aspiration of the immature oocyte from
ovarian follicles .
Additionally, metaphase II stage oocytes, which have been matured in vivo have
been successfully used in nuclear transfer techniques. Essentially, mature metaphase II
oocytes are collected surgically from either non-superovulated or superovulated cows or
heifers 35 to 48 hours past the onset of estrus or past the injection of human chorionic
gonadotropin (hCG) or similar hormone. The stage of maturation of the oocyte at enucleation and nuclear transfer has been
reported to be significant to the success of NT methods. (See e.g., Prather et al.,
Differentiation, 48, 1-8, 1991). In general, previous successful mammalian embryo
cloning practices used metaphase II stage oocyte as the recipient oocyte because at this
stage it is believed that the oocyte can be or is sufficiently "activated" to treat the intro¬
duced nucleus as it does a fertilizing sperm. In domestic animals, and especially cattle,
the oocyte activation period generally ranges from about 16-52 hours, preferably about
28-42 hours post-aspiration.
For example, immature oocytes may be washed in HEPES buffered hamster
embryo culture medium (HECM) as described in Seshagine et al., Biol. Reprod. , 40, 544-
606, 1989, and then placed into drops of maturation medium consisting of 50 microliters
of tissue culture medium (TCM) 199 containing 10% fetal calf serum which contains
appropriate gonadotropins such as luteinizing hormone (LH) and follicle stimulating
hormone (FSH), and estradiol under a layer of lightweight paraffin or silicon at 39°C.
After a fixed time maturation period, which typically will range from about 10 to
40 hours, and preferably about 16-18 hours, the oocytes will typically be enucleated.
Prior to enucleation the oocytes will preferably be removed and placed in HECM
containing 1 milligram per milliliter of hyaluronidase prior to removal of cumulus cells.
This may be effected by repeated pipetting through very fine bore pipettes or by vortexing
briefly. The stripped oocytes are then screened for polar bodies, and the selected
metaphase II oocytes, as determined by the presence of polar bodies, are then used for nuclear transfer. Enucleation follows. As noted above, enucleation may be effected
before or after introduction of donor cell or nucleus because the donor nucleus is readily
discernible from endogenous nucleus.
Enucleation may be effected by known methods, such as described in U.S. Patent
No. 4,994,384 which is incorporated by reference herein. For example, metaphase II
oocytes are either placed in HECM, optionally containing 7.5 micrograms per milliliter
cytochalasin B, for immediate enucleation, or may be placed in a suitable medium, for
example CRlaa, plus 10% estrus cow serum, and then enucleated later, preferably not
more than 24 hours later, and more preferably 16-18 hours later.
Enucleation may be accomplished microsurgically using a micropipette to remove
the polar body and the adjacent cytoplasm. The oocytes may then be screened to identify
those of which have been successfully enucleated. This screening may be effected by
staining the oocytes with 1 microgram per milliliter 33342 Hoechst dye in HECM, and
then viewing the oocytes under ultraviolet irradiation for less than 10 seconds. The
oocytes that have been successfully enucleated can then be placed in a suitable culture
In the present invention, the recipient oocytes will typically be enucleated at a time
ranging from about 10 hours to about 40 hours after the initiation of in vitro maturation,
more preferably from about 16 hours to about 24 hours after initiation of in vitro matura-
tion, and most preferably about 16-18 hours after initiation of in vitro maturation. Enucleation may be effected before, simultaneous or after nuclear transfer. Also,
enucleation may be effected before, after or simultaneous to activation.
A single animal or human cell or nucleus derived therefrom which is typically
heterologous to the enucleated oocyte will then be transferred into the perivitelline space
of the oocyte, typically enucleated, used to produce the NT unit. However, removal of
endogenous nucleus may alternatively be effected after nuclear transfer. The animal or
human cell or nucleus and the enucleated oocyte will be used to produce NT units ac¬
cording to methods known in the art. For example, the cells may be fused by electro-
fusion. Electrofusion is accomplished by providing a pulse of electricity that is sufficient
to cause a transient break down of the plasma membrane. This breakdown of the plasma
membrane is very short because the membrane reforms rapidly. Essentially, if two
adjacent membranes are induced to break down, upon reformation the lipid bilayers
intermingle and small channels will open between the two cells. Due to the ther-
modynamic instability of such a small opening, it enlarges until the two cells become one.
Reference is made to U.S. Patent 4,997,384, by Prather et al., (incorporated by reference
in its entirety herein) for a further discussion of this process. A variety of electrofusion
media can be used including e.g., sucrose, mannitol, sorbitol and phosphate buffered
solution. Fusion can also be accomplished using Sendai virus as a fusogenic agent
(Graham, Wister lnot. Symp. Monogr., 9, 19, 1969).
Also, in some cases (e.g. with small donor nuclei) it may be preferable to inject the
nucleus directly into the oocyte rather than using electroporation fusion. Such techniques are disclosed in Collas and Barnes, Mol. Reprod. Dev., 38:264-267 (1994), and
incorporated by reference in its entirety herein.
Preferably, the human or animal cell and oocyte are electrofused in a 500 μm
chamber by application of an electrical pulse of 90- 120V for about 15 μsec, about 24
hours after initiation of oocyte maturation. After fusion, the resultant fused NT units are
preferably placed in a suitable medium until activation, e.g., one identified infra. -
Typically activation will be effected shortly thereafter, typically less than 24 hours later,
and preferably about 4-9 hours later. However, it is also possible to activate the recipient
oocyte before or proximate (simultaneous) to nuclear transfer, which may or may not be
enucleated. For example, activation may be effected from about twelve hours prior to
nuclear transfer to about twenty-four hours after nuclear transfer. More typically,
activation is effected simultaneous or shortly after nuclear transfer, e.g., about four to nine
The NT unit may be activated by known methods. Such methods include, e.g.,
culturing the NT unit at sub-physiological temperature, in essence by applying a cold, or
actually cool temperature shock to the NT unit. This may be most conveniently done by
culturing the NT unit at room temperature, which is cold relative to the physiological
temperature conditions to which embryos are normally exposed.
Alternatively, activation may be achieved by application of known activation
agents. For example, penetration of oocytes by sperm during fertilization has been shown
to activate prefusion oocytes to yield greater numbers of viable pregnancies and multiple genetically identical calves after nuclear transfer. Also, treatments such as electrical and
chemical shock or cycloheximide treatment may also be used to activate NT embryos
after fusion. Suitable oocyte activation methods are the subject of U.S. Patent No.
5,496,720, to Susko-Parrish et al., which is herein incorporated by reference.
5 For example, oocyte activation may be effected by simultaneously or sequentially:
(i) increasing levels of divalent cations in the oocyte, and
(ii) reducing phosphorylation of cellular proteins in the oocyte.
This will generally be effected by introducing divalent cations into the oocyte
cytoplasm, e.g., magnesium, strontium, barium or calcium, e.g., in the form of an iono-
0 phore. Other methods of increasing divalent cation levels include the use of electric
shock, treatment with ethanol and treatment with caged chelators.
Phosphorylation may be reduced by known methods, e.g., by the addition of kinase
inhibitors, e.g., serine-threonine kinase inhibitors, such as 6-dimethylamino-purine,
staurosporine, 2-arninopurine, and sphingosine.
5 Alternatively, phosphorylation of cellular proteins may be inhibited by
introduction of a phosphatase into the oocyte, e.g., phosphatase 2A and phosphatase 2B.
Specific examples of activation methods are listed below.
1. Activation by Ionomycin and DMAP
1- Place oocytes in Ionomycin (5 μM) with 2 mM of DMAP for 4
o minutes; 2- Move the oocytes into culture media with 2 mM of DMAP for 4
3- Rinse four times and place in culture.
2. Activation by Ionomycin DMAP and Roscovitin
1- Place oocytes in Ionomycin (5 μM) with 2 mM of DMAP for four
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
1- Place oocytes in Ionomycin (5 microM) for four minutes;
2- Move oocytes to culture media containing 5 μg/ml of cytochalasin
B and 5 μg/ml of cycloheximide for five hours;
3- Rinse four times and place in culture.
4. Activation by electrical pulses
1- Place eggs in mannitol media containing 100 μM CaCL2;
2- Deliver three pulses of 1.0 kVcm"1 for 20 μsec, each pulse 22
3- Move oocytes to culture media containing 5 μg/ml of cytochalasin
B for three hours. 5. Activation by exposure with ethanol followed by cytochalasin and
1- Place oocytes in 7% ethanol for one minute;
2- Move oocytes to culture media containing 5 μg/ml of cytochalasin
B and 5 μg/ml of cycloheximide for five hours;
3- Rinse four times and place in culture.
6. Activation by microinjection of adenophostine
1- Inject oocytes with 10 to 12 picoliters of a solution containing 10
μM of adenophostine;
2- Put oocytes in culture.
7. Activation by microinjection of sperm factor
1 - Inject oocytes with 10 to 12 picoliters of sperm factor isolated, e.g.,
from primates, pigs, bovine, sheep, goats, horses, mice, rats, rabbits
2- Put eggs in culture.
8. Activation by microinjection of recombinant sperm factor.
9. Activation by Exposure to DMAP followed by Cycloheximide and Cytochalasin B
Place oocytes or NT units, typically about 22 to 28 hours post
maturation in about 2 mM DMAP for about one hour, followed by incubation for about two to twelve hours, preferably about eight hours, in
5 μg/ml of cytochalasin B and 20 μg/ml cycloheximide.
The above activation protocols are exemplary of protocols used for nuclear transfer
procedures, e.g., those including the use of primate or human donor cells or oocytes.
However, the above activation protocols may be used when either or both the donor cell
and nucleus is of ungulate origin, e.g., a sheep, buffalo, horse, goat, bovine, pig and/or
wherein the oocyte is of ungulate origin, e.g., sheet, pig, buffalo, horse, goat, bovine, etc.,
as well as for other species.
As noted, activation may be effected before, simultaneous, or after nuclear
transfer. In general, activation will be effected about 40 hours prior to nuclear transfer
and fusion to about 40 hours after nuclear transfer and fusion, more preferably about 24
hours before to about 24 hours after nuclear transfer and fusion, and most preferably from
about 4 to 9 hours before nuclear transfer and fusion to about 4 to 9 hours after nuclear
transfer and fusion. Activation is preferably effected after or proximate to in vitro or in
vivo maturation of the oocyte, e.g., approximately simultaneous or within about 40 hours
of maturation, more preferably within about 24 hours of maturation.
Activated NT units may be cultured in a suitable in vitro culture medium until the
generation of embryonic or stem-like cells and cell colonies. Culture media suitable for
culturing and maturation of embryos are well known in the art. Examples of known
media, which may be used for bovine embryo culture and maintenance, include Ham's
F-10 + 10% fetal calf serum (FCS), Tissue Culture Medium- 199 (TCM-199) + 10% fetal calf serum, Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's Phosphate Buffered
Saline (PBS), Eagle's and Whitten's media. One of the most common media used for the
collection and maturation of oocytes is TCM-199, and 1 to 20% serum supplement
including fetal calf serum, newborn serum, estrual cow serum, lamb serum or steer
serum. A preferred maintenance medium includes TCM-199 with Earl salts, 10% fetal
calf serum, 0.2 Ma pyruvate and 50 μg/ml gentamicin sulphate. Any of the above may
also involve co-culture with a variety of cell types such as granulosa cells, oviduct cells,
BRL cells and uterine cells and STO cells.
In particular, human epithelial cells of the endometrium secrete leukemia
inhibitory factor (LIF) during the preimplantation and implantation period. Therefore,
the addition of LIF to the culture medium could be of importance in enhancing the in
vitro development of the reconstructed embryos. The use of LIF for embryonic or stem¬
like cell cultures has been described in U.S. Patent 5,712,156, which is herein
incorporated by reference.
Another maintenance medium is described in U.S. Patent 5,096,822 to Rosenkrans,
Jr. et al., which is incorporated herein by reference. This embryo medium, named CR1,
contains the nutritional substances necessary to support an embryo. CR1 contains
hemicalcium L-lactate in amounts ranging from 1.0 mM to 10 mM, preferably 1.0 mM
to 5.0 mM. Hemicalcium L-lactate is L-lactate with a hemicalcium salt incorporated
thereon. Also, suitable culture medium for maintaining human embryonic cells in culture
as discussed in Thomson et al., Science, 282:1145-1147 (1998) and Proc. Nαtl. Acαd. Sci,
USA, 92:7844-7848 (1995).
Afterward, the cultured NT unit or units are preferably washed and then placed in
a suitable media, e.g., CRIaa medium, Ham's F-10, Tissue Culture Media -199 (TCM-
199). Tyrodes-Albumin-Lactate-Pyruvate (TALP) Dulbecco's Phosphate Buffered Saline
(PBS), Eagle's or Whitten's, preferably containing about 10% FCS. Such culturing will
preferably be effected in well plates which contain a suitable confluent feeder layer.
Suitable feeder layers include, by way of example, fibroblasts and epithelial cells, e.g.,
fibroblasts and uterine epithelial cells derived from ungulates, chicken fibroblasts, murine
(e.g., mouse or rat) fibroblasts, STO and SI-m220 feeder cell lines, and BRL cells.
In the preferred embodiment, the feeder cells will comprise mouse embryonic
fibroblasts. Means for preparation of a suitable fibroblast feeder layer are described in
the example which follows and is well within the skill of the ordinary artisan.
The NT units are cultured on the feeder layer until the NT units reach a size
suitable for obtaining cells which may be used to produce embryonic stem-like cells or
cell colonies. Preferably, these NT units will be cultured until they reach a size of at least
about 2 to 400 cells, more preferably about 4 to 128 cells, and most preferably at least
about 50 cells. The culturing will be effected under suitable conditions, i.e., about 38.5 °C
and 5% C02, with the culture medium changed in order to optimize growth typically
about every 2-5 days, preferably about every 3 days. In the case of human cell/enucleated bovine oocyte derived NT units, sufficient
cells to produce an ES cell colony, typically on the order of about 50 cells, will be
obtained about 12 days after initiation of oocyte activation. However, this may vary
dependent upon the particular cell used as the nuclear donor, the species of the particular
oocyte, and culturing conditions. One skilled in the art can readily ascertain visually
when a desired sufficient number of cells has been obtained based on the morphology of
the cultured NT units.
In the case of human/human nuclear transfer embryos, or other embryos produced
using non-human primate donor or oocyte, it may be advantageous to use culture medium
known to be useful for maintaining human and other primate cells in tissue culture.
Examples of a culture media suitable for human embryo culture include the medium
reported in Jones et al, Human Reprod., 13(1):169-177 (1998), the Pl-catalog #99242
medium, and the P-l catalog #99292 medium, both available from Irvine Scientific, Santa
Ana, California, and those used by Thomson et al. (1998) and (1995), which references
are incorporated by reference in their entirety.
Another preferred medium comprises ACM + uridine + glucose + 1000 IU of LIF.
As discussed above, the cells used in the present invention will preferably
comprise mammalian somatic cells, most preferably cells derived from an actively
proliferating (non-quiescent) mammalian cell culture. In an especially preferred
embodiment, the donor cell will be genetically modified by the addition, deletion or
substitution of a desired DNA sequence. For example, the donor cell, e.g., a keratinocyte or fibroblast, e.g., of human, primate or bovine origin, may be transfected or transformed
with a DNA construct that provides for the expression of a desired gene product, e.g.,
therapeutic polypeptide. Examples thereof include lymphokines, e.g., IGF-I, IGF-II,
interferons, colony stimulating factors, connective tissue polypeptides such as collagens,
genetic factors, clotting factors, enzymes, enzyme inhibitors, etc.
Also, as discussed above, the donor cells may be modified prior to nuclear transfer,
e.g., to effect impaired cell lineage development, enhanced embryonic development
and/or inhibition of apoptosis. Examples of desirable modifications are discussed further
One aspect of the invention will involve genetic modification of the donor cell,
e.g., a human cell, such that it is lineage deficient and therefore when used for nuclear
transfer it will be unable to give rise to a viable offspring. This is desirable especially in
the context of human nuclear transfer embryos, wherein for ethical reasons, production
of a viable embryo may be an unwanted outcome. This can be effected by genetically
engineering a human cell such that it is incapable of differentiating into specific cell
lineages when used for nuclear transfer. In particular, cells may be genetically modified
such that when used as nuclear transfer donors the resultant "embryos" do not contain or
substantially lack at least one of mesoderm, endoderm or ectoderm tissue.
This can be accomplished by, e.g., knocking out or impairing the expression of one
or more mesoderm, endoderm or ectoderm specific genes. Examples thereof include:
Mesoderm: SRF, MESP- 1 , HNF-4, beta-I integrin, MSD ; Endoderm: GATA-6, GATA-4;
Ectoderm: RNA helicase A, H beta 58.
The above list is intended to be exemplary and non-exhaustive of known genes
which are involved in the development of mesoderm, endoderm and ectoderm. The
generation of mesoderm deficient, endoderm deficient and ectoderm deficient cells and
embryos has been previously reported in the literature. See, e.g., Arsenian et al, EMBO
J., Vol. 17(2):6289-6299 (1998); Saga Y, Mech. Dev., Vol. 75(l-2):53-66 (1998);
Holdener et al, Development, Vol. 120(5): 1355-1346 (1994); Chen et al, Genes Dev. Vol.
8(20):2466-2477 (1994); Rohwedel et al, Dev. Biol., 201(2):167-189 (1998) (mesoderm);
Morrisey et al, Genes, Dev., Vol. 12(22):3579-3590 (1998); Soudais et al, Development,
Vol. 121(11):3877-3888 (1995) (endoderm); and Lee et al, Proc. Natl. Acad. Sci. USA,
Vol. 95:(23):13709-13713 (1998); and Radice et al, Development, Vol. 111(3):801-811
In general, a desired somatic cell, e.g., a human keratinocyte, epithelial cell or
fibroblast, will be genetically engineered such that one or more genes specific to
particular cell lineages are "knocked out" and/or the expression of such genes
significantly impaired. This may be effected by known methods, e.g., homologous
recombination. A preferred genetic system for effecting "knock-out" of desired genes is
disclosed by Capecchi et al, U.S. Patents 5,631,153 and 5,464,764, which reports
positive-negative selection (PNS) vectors that enable targeted modification of DNA
sequences in a desired mammalian genome. Such genetic modification will result in a cell that is incapable of differentiating into a particular cell lineage when used as a nuclear
This genetically modified cell will be used to produce a lineage-defective nuclear
transfer embryo, i.e., that does not develop at least one of a functional mesoderm,
endoderm or ectoderm. Thereby, the resultant embryos, even if implanted, e.g., into a
human uterus, would not give rise to a viable offspring. However, the ES cells that result
from such nuclear transfer will still be useful in that they will produce cells of the one or
two remaining non-impaired lineage. For example, an ectoderm deficient human nuclear
transfer embryo will still give rise to mesoderm and endoderm derived differentiated
cells. An ectoderm deficient cell can be produced by deletion and/or impairment of one
or both of RNA helicase A or H beta 58 genes.
These lineage deficient donor cells may also be genetically modified to express
another desired DNA sequence.
Thus, the genetically modified donor cell will give rise to a lineage-deficient
blastocyst which, when plated, will differentiate into at most two of the embryonic germ
Alternatively, the donor cell can be modified such that it is "mortal". This can be
achieved by expressing anti-sense or ribozyme telomerase genes. This can be effected
by known genetic methods that will provide for expression of antisense DNA or
ribozymes, or by gene knockout. These "mortal" cells, when used for nuclear transfer,
will not be capable of differentiating into viable offspring. Another preferred embodiment of the present invention is the production of
nuclear transfer embryos that grow more efficiently in tissue culture. This is
advantageous in that it should reduce the requisite time and necessary fusions to produce
ES cells and/or offspring (if the blastocysts are to be implanted into a female surrogate).
This is desirable also because it has been observed that blastocysts and ES cells resulting
from nuclear transfer may have impaired development potential. While these problems
may often be alleviated by alteration of tissue culture conditions, an alternative solution
is to enhance embryonic development by enhancing expression of genes involved in
For example, it has been reported that the gene products of the Ped type, which are
members of the MHC I family, are of significant importance to embryonic development.
More specifically, it has been reported in the case of mouse preimplantation embryos that
the Q7 and Q9 genes are responsible for the "fast growth" phenotype. Therefore, it is
anticipated that introduction of DNAs that provide for the expression of these and related
genes, or their human or other mammalian counterparts into donor cells, will give rise to
nuclear fransfer embryos that grow more quickly. This is particularly desirable in the
context of cross-species nuclear transfer embryos which may develop less efficiently in
tissue culture than nuclear transfer embryos produced by fusion of cells or nuclei of the
In particular, a DNA construct containing the Q7 and/or Q9 gene will be
introduced into donor somatic cells prior to nuclear transfer. For example, an expression construct can be constructed containing a strong constitutive mammalian promoter
operably linked to the Q7 and or Q9 genes, an IRES, one or more suitable selectable
markers, e.g,. neomycin, ADA, DHFR, and a poly-A sequence, e.g., bGH polyA
sequence. Also, it may be advantageous to further enhance Q7 and Q9 gene expression
by the inclusion of insulates. It is anticipated that these genes will be expressed early on
in blastocyst development as these genes are highly conserved in different species, e.g.,
bo vines, goats, porcine, dogs, cats, and humans. Also, it is anticipated that donor cells
can be engineered to affect other genes that enhance embryonic development. Thus, these
genetically modified donor cells should produce blastocysts and preimplantation stage
embryos more efficiently.
Still another aspect of the invention involves the construction of donor cells that
are resistant to apoptosis, i.e., programmed cell death. It has been reported in the
literature that cell death related genes are present in preimplantation stage embryos.
(Adams et al, Science, 281(5381): 1322-1326 (1998)). Genes reported to induce apoptosis
include, e.g., Bad, Bok, BH3, Bik, Hrk, BNIP3, 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, LMW5-HL, ORF16, Ks-Bel-2,
E1B-19K, and CED-9.
Thus, donor cells can be constructed wherein genes that induce apoptosis are
"knocked out" or wherein the expression of genes that protect the cells from apoptosis is
enhanced or turned on during embryonic development. For example, this can be effected by introducing a DNA construct that provides
for regulated expression of such protective genes, e.g., Bcl-2 or related genes during
embryonic development. Thereby, the gene can be "turned on" by culturing the embryo
under specific growth conditions. Alternatively, it can be linked to a constitutive
More specifically, a DNA construct containing a Bcl-2 gene operably linked to a
regulatable or constitutive promoter, e.g., PGK, SV40, CMV, ubiquitin, or beta-actin, an
IRES, a suitable selectable marker, and a poly-A sequence can be constructed and
introduced into a desired donor mammalian cell, e.g., human keratinocyte or fibroblast.
These donor cells, when used to produce nuclear transfer embryos, should be
resistant to apoptosis and thereby differentiate more efficiently in tissue culture. Thereby,
the speed and/or number of suitable preimplantation embryos produced by nuclear
transfer can be increased.
Another means of accomplishing the same result is to impair the expression of one
or more genes that induce apoptosis. This will be effected by knock-out or by the use of
antisense or ribozymes against genes that are expressed in and which induce apoptosis
early on in embryonic development. Examples thereof are identified above. Cell death
genes that may be expressed in the antisense orientation include BAX, Apaf-1, and
capsases. Additionally, a transgene may be introduced that encodes for methylase or
demethylase in the sense or antisense orientation. DNAs that encode methylase and
demethylase enzymes are well known in the art. Still alternatively, donor cells may be constructed containing both modifications, i.e., impairment of apoptosis-inducing genes
and enhanced expression of genes that impede or prevent apoptosis. The construction and
selection of genes that affect apoptosis, and cell lines that express such genes, is disclosed
in U.S. Patent No. 5,646,008, which patent is incorporated by reference herein. Many
DNAs that promote or inhibit apoptosis have been reported and are the subject of
Another means of enhancing cloning efficiency is to select cells of a particular cell
cycle stage as the donor cell. It has been reported that this can have significant effects on
nuclear transfer efficiency. (Barnes et al, Mol. Reprod. Devel., 36(1):33-41 (1993).
Different methods for selecting cells of a particular cell cycle stage have been reported
and include serum starvation (Campbell et al, Nature, 380:64-66 (1996); Wilmut et al,
Nature, 385:810-813 (1997), and chemical synchronization (Urbani et al, Exp. Cell Res.,
219(1):159-168 (1995). For example, a particular cyclin DNA may be operably linked
to a regulatory sequence, together with a detectable marker, e.g., green fluorescent protein
(GFP), followed by the cyclin destruction box, and optionally insulation sequences to
enhance cyclin and marker protein expression. Thereby, cells of a desired cell cycle can
be easily visually detected and selected for use as a nuclear transfer donor. An example
thereof is the cyclin Dl gene in order to select for cells that are in Gl. However, any
cyclin gene should be suitable for use in the claimed invention. (See, e.g., King et al,
Mol. Biol. Cell, Vol. 7(9):1343-1357 (1996)). However, a less invasive or more efficient method for producing cells of a desired
cell cycle stage are needed. It is anticipated that this can be effected by genetically
modifying donor cells such that they express specific cyclins under detectable conditions.
Thereby, cells of a specific cell cycle can be readily discerned from other cell cycles.
Cyclins are proteins that are expressed only during specific stages of the cell cycle.
They include cyclin Dl, D2 and D3 in Gl phase, cyclin Bl and B2 in G2/M phase and
cyclin E, A and H in S phase. These proteins are easily translated and destroyed in the
cytogolcytosol. This "transient" expression of such proteins is attributable in part to the
presence of a "destruction box", which is a short amino acid sequence that is part of the
protein that functions as a tag to direct the prompt destruction of these proteins via the
ubiquitin pathway. (Adams et al, Science, 281 (5321): 1322-1326 (1998)).
In the present invention, donor cells will be constructed that express one or more
of such cyclin genes under easily detectable conditions, preferably visualizable, e.g., by
the use of a fluorescent label. For example, a particular cyclin DNA may be operably
linked to a regulatory sequence, together with a detectable marker, e.g., green fluorescent
protein (GFP), followed by the cyclin destruction box, and optionally insulation
sequences to enhance cyclin and/or marker protein expression. Thereby, cells of a
desired cell cycle can be easily visually detected and selected for use as a nuclear transfer
donor. An example thereof is the cyclin Dl gene which can be used to select for cells that
are in Gl . However, any cyclin gene should be suitable for use in the claimed invention.
(See, e.g., King et al, Mol. Biol. Cell, Vol. 7(9):1343-1357 (1996)). As discussed, the present invention provides different methods for enhancing
nuclear transfer efficiency, preferably a cross-species nuclear transfer process. While the
present inventors have demonstrated that nuclei or cells of one species when inserted or
fused with an enucleated oocyte of a different species can give rise to nuclear transfer
embryos that produce blastocysts, which embryos can give rise to ES cell lines, the
efficiency of such process is quite low. Therefore, many fusions typically need to be
effected to produce a blastocyst the cells of which may be cultured to produce ES cells
and ES cell lines. Yet another means for enhancing the development of nuclear transfer
embryos in vitro is by optimizing culture conditions. One means of achieving this result
will be to culture NT embryos under conditions impede apoptosis. With respect to this
embodiment of the invention, it has been found that proteases such as capsases can cause
oocyte death by apoptosis similar to other cell types. (See, Jurisicosva et al, Mol.
Reprod. Devel, 51(3):243-253 (1998).)
It is anticipated that blastocyst development will be enhanced by including in
culture media used for nuclear transfer and to maintain blastocysts or culture
preimplantation stage embryos one or more capsase inhibitors. Such inhibitors include
by way of example capsase-4 inhibitor I, capsase-3 inhibitor I, capsase-6 inhibitor II,
capsase-9 inhibitor II, and capsase- 1 inhibitor I. The amount thereof will be an amount
effective to inhibit apoptosis, e.g., 0.00001 to 5.0% by weight of medium; more
preferably 0.01% to 1.0% by weight of medium. Thus, the foregoing methods may be used to increase the efficiency of nuclear transfer by enhancing subsequent blastocyst and
embryo development in tissue culture.
After NT units of the desired size are obtained, the cells are mechanically removed
from the zone and are then used to produce embryonic or stem-like cells and cell lines.
This is preferably effected by taking the clump of cells which comprise the NT unit,
which typically will contain at least about 50 cells, washing such cells, and plating the
cells onto a feeder layer, e.g., irradiated fibroblast cells. Typically, the cells used to
obtain the stem-like cells or cell colonies will be obtained from the inner most portion of
the cultured NT unit which is preferably at least 50 cells in size. However, NT units of
smaller or greater cell numbers as well as cells from other portions of the NT unit may
also be used to obtain ES-like cells and cell colonies.
It is further envisioned that a longer exposure of donor cell DNA to the oocyte 's
cytosol may facilitate the dedifferentiation process. This can be accomplished by re-
cloning, i.e., by taking blastomeres from a reconstructed embryo and fusing them with
a new enucleated oocyte. Alternatively, the donor cell may be fused with an enucleated
oocyte and four to six hours later, without activation, chromosomes removed and fused
with a younger oocyte. Activation would occur thereafter.
The cells are maintained in the feeder layer in a suitable growth medium, e.g.,
alpha MEM supplemented with 10% FCS and 0.1 mM beta-mercaptoethanol (Sigma) and
L-glutamine. The growth medium is changed as often as necessary to optimize growth,
e.g., about every 2-3 days. This culturing process results in the formation of embryonic or stem-like cells or
cell lines. In the case of human cell/bovine oocyte derived NT embryos, colonies are
observed by about the second day of culturing in the alpha MEM medium. However, this
time may vary dependent upon the particular nuclear donor cell, specific oocyte and
culturing conditions. One skilled in the art can vary the culturing conditions as desired
to optimize growth of the particular embryonic or stem-like cells. Other suitable media
are disclosed herein.
The embryonic or stem-like cells and cell colonies obtained will typically exhibit
an appearance similar to embryonic or stem-like cells of the species used as the nuclear
cell donor rather than the species of the donor oocyte. For example, in the case of embry¬
onic or stem-like cells obtained by the fransfer of a human nuclear donor cell into an
enucleated bovine oocyte, the cells exhibit a morphology more similar to mouse
embryonic stem cells than bovine ES-like cells.
More specifically, the individual cells of the human ES-line cell colony are not
well defined, and the perimeter of the colony is refractive and smooth in appearance.
Further, the cell colony has a longer cell doubling time, about twice that of mouse ES
cells. Also, unlike bovine and porcine derived ES cells, the colony does not possess an
As discussed above, it has been reported by Thomson, in U.S. Patent 5,843,780,
that primate stem cells are SSEA-1 (-), SSEA-4 (+), TRA-1-60 (+), TRA-1-81 (+) and alkaline phosphatase (+). It is anticipated that human and primate ES cells produced
according to the present methods will exhibit similar or identical marker expression.
Alternatively, that such cells are actual human or primate embryonic stem cells
will be confirmed based on their capability of giving rise to all of mesoderm, ectoderm
and endoderm tissues. This will be demonstrated by culturing ES cells produced
according to the invention under appropriate conditions, e.g., as disclosed by Thomsen,
U.S. Patent 5,843,780, incorporated by reference in its entirety herein. Alternatively, the
fact that the cells produced according to the invention are pluripotent will be confirmed
by injecting such cells into an animal, e.g., a SCID mouse, or large agricultural animal,
and thereafter obtaining tissues that result from said implanted cells. These implanted ES
cells should give rise to all different types of differentiated tissues, i.e., mesoderm,
ectoderm, and endodermal tissues.
The resultant embryonic or stem-like cells and cell lines, preferably human
embryonic or stem-like cells and cell lines, have numerous therapeutic and diagnostic
applications. Most especially, such embryonic or stem-like cells may be used for cell
transplantation therapies. Human embryonic or stem-like cells have application in the
treatment of numerous disease conditions.
Still another object of the present invention is to improve the efficacy of nuclear
transfer, e.g., cross-species nuclear transfer by introducing mitochondrial DNA of the
same species as the donor cell or nucleus into the recipient oocyte before or after nuclear
transfer, before or after activation, and before or after fusion and cleavage. Preferably, if the donor cell is human, human mitochondrial DNA will be derived from cells of the
particular donor, e.g., liver cells and tissue.
Methods for isolating mitochondria are well known in the art. Mitochondria can
be isolated from cells in tissue culture, or from tissue. The particular cells or tissue will
depend upon the particular species of the donor cell. Examples of cells or tissues that
may be used as sources of mitochondria include fibroblasts, epithelium, liver, lung,
keratinocyte, stomach, heart, bladder, pancreas, esophageal, lymphocytes, monocytes,
mononuclear cells, cumulus cells, uterine cells, placental cells, intestinal cells,
hematopoietic cells, and tissues containing such cells.
For example, mitochondria can be isolated from tissue culture cells and rat liver.
It is anticipated that the same or similar procedures may be used to isolate mitochondria
from other cells and tissues. As noted above, preferred source of mitochondria comprises
human liver tissue because such cells contain a large number of mitochondria. Those
skilled in the art will be able to modify the procedure as necessary, dependent upon the
particular cell line or tissue. The isolated DNA can also be further purified, if desired,
known methods, e.g., density gradient centrifugation.
In this regard, it is known that mouse embryonic stem (ES) cells are capable of
differentiating into almost any cell type, e.g., hematopoietic stem cells. Therefore, human
embryonic or stem-like cells produced according to the invention should possess similar
differentiation capacity. The embryonic or stem-like cells according to the invention will
be induced to differentiate to obtain the desired cell types according to known methods. For example, the subject human embryonic or stem-like cells may be induced to
differentiate into hematopoietic stem cells, muscle cells, cardiac muscle cells, liver cells,
cartilage cells, epithelial cells, urinary tract cells, etc., by culturing such cells in
differentiation medium and under conditions which provide for cell differentiation.
Medium and methods which result in the differentiation of embryonic stem cells are
known in the art as are suitable culturing conditions.
For example, Palacios et al, Proc. Natl. Acad. Sci, USA, 92:7530-7537 (1995)
teaches the production of hematopoietic stem cells from an embryonic cell line by
subjecting stem cells to an induction procedure comprising initially culturing aggregates
of such cells in a suspension culture medium lacking retinoic acid followed by culturing
in the same medium containing retinoic acid, followed by transferral of cell aggregates
to a substrate which provides for cell attachment.
Moreover, Pedersen, J. Reprod. Fertil. Dev., 6:543-552 (1994) is a review article
which references numerous articles disclosing methods for in vitro differentiation of
embryonic stem cells to produce various differentiated cell types including hematopoietic
cells, muscle, cardiac muscle, nerve cells, among others.
Further, Bain et al, Dev. Biol, 168:342-357 (1995) teaches in vitro differentiation
of embryonic stem cells to produce neural cells which possess neuronal properties. These
references are exemplary of reported methods for obtaining differentiated cells from
embryonic or stem-like cells. These references and in particular the disclosures therein relating to methods for differentiating embryonic stem cells are incorporated by reference
in their entirety herein.
Thus, using known methods and culture medium, one skilled in the art may culture
the subject embryonic or stem-like cells to obtain desired differentiated cell types, e.g.,
neural cells, muscle cells, hematopoietic cells, etc. In addition, the use of inducible Bcl-2
or Bcl-xl might be useful for enhancing in vitro development of specific cell lineages.
In vivo, Bcl-2 prevents many, but not all, forms of apoptotic cell death that occur during
lymphoid and neural development. A thorough discussion of how Bcl-2 expression might
be used to inhibit apoptosis of relevant cell lineages following transfection of donor cells
is disclosed in U.S. Patent No. 5,646,008, which is herein incorporated by reference.
The subject embryonic or stem-like cells may be used to obtain any desired
differentiated cell type. Therapeutic usages of such differentiated human cells are
unparalleled. For example, human hematopoietic stem cells may be used in medical
treatments requiring bone marrow transplantation. Such procedures are used to treat
many diseases, e.g., late stage cancers such as ovarian cancer and leukemia, as well as
diseases that compromise the immune system, such as AIDS. Hematopoietic stem cells
can be obtained, e.g., by fusing adult somatic cells of a cancer or AIDS patient, e.g.,
epithelial cells or lymphocytes with an enucleated oocyte, e.g., bovine oocyte, obtaining
embryonic or stem-like cells as described above, and culturing such cells under conditions which favor differentiation, until hematopoietic stem cells are obtained. Such
hematopoietic cells may be used in the treatment of diseases including cancer and AIDS.
Alternatively, adult somatic cells from a patient with a neurological disorder may
be fused with an enucleated animal oocyte, e.g., a primate or bovine oocyte, human
embryonic or stem-like cells obtained therefrom, and such cells cultured under
differentiation conditions to produce neural cell lines. Specific diseases treatable by
transplantation of such human neural cells include, by way of example, Parkinson's
disease, Alzheimer's disease, ALS and cerebral palsy, among others. In the specific case
of Parkinson's disease, it has been demonstrated that transplanted fetal brain neural cells
make the proper connections with surrounding cells and produce dopamine. This can
result in long-term reversal of Parkinson's disease symptoms.
To allow for specific selection of differentiated cells, donor cells may be
transfected with selectable markers expressed via inducible promoters, thereby permitting
selection or enrichment of particular cell lineages when differentiation is induced. For
example, CD34-neo may be used for selection of hematopoietic cells, Pwl-neo for
muscle cells, Mash-1-neo for sympathetic neurons, Mal-neo for human CNS neurons of
the grey matter of the cerebral cortex, etc.
The great advantage of the subject invention is that it provides an essentially
limitless supply of isogenic or synegenic human cells suitable for transplantation.
Therefore, it will obviate the significant problem associated with current transplantation
methods, i.e., rejection of the transplanted tissue which may occur because of host-vs- graft or graft-vs-host rejection. Conventionally, rejection is prevented or reduced by the
administration of anti-rejection drugs such as cyclosporin. However, such drugs have
significant adverse side-effects, e.g., immunosuppression, carcinogenic properties, as well
as being very expensive. The present invention should eliminate, or at least greatly
reduce, the need for anti-rejection drugs, such as cyclosporine, imulan, FK-506,
glucocorticoids, and rapamycin, and derivatives thereof.
Other diseases and conditions treatable by isogenic cell therapy include, by way
of example, spinal cord injuries, multiple sclerosis, muscular dystrophy, diabetes, liver
diseases, i.e., hypercholesterolemia, heart diseases, cartilage replacement, burns, foot
ulcers, gastrointestinal diseases, vascular diseases, kidney disease, urinary tract disease,
and aging related diseases and conditions.
Also, human embryonic or stem-like cells produced according to the invention
may be used to produce genetically engineered or transgenic human differentiated cells.
Essentially, this will be effected by introducing a desired gene or genes, which may be
heterologous, or removing all or part of an endogenous gene or genes of human
embryonic or stem-like cells produced according to the invention, and allowing such cells
to differentiate into the desired cell type. A preferred method for achieving such
modification is by homologous recombination because such technique can be used to
insert, delete or modify a gene or genes at a specific site or sites in the stem-like cell
genome. This methodology can be used to replace defective genes, e.g., defective immune
system genes, cystic fibrosis genes, or to infroduce genes which result in the expression
of therapeutically beneficial proteins such as growth factors, lymphokines, cytokines,
enzymes, etc. For example, the gene encoding brain derived growth factor may be
infroduced into human embryonic or stem-like cells, the cells differentiated into neural
cells and the cells transplanted into a Parkinson's patient to retard the loss of neural cells
during such disease.
Previously, cell types transfected with BDNF varied from primary cells to
immortalized cell lines, either neural or non-neural (myoblast and fibroblast) derived
cells. For example, asfrocytes have been transfected with BDNF gene using retro viral
vectors, and the cells grafted into a rat model of Parkinson's disease (Yoshimoto et al.,
Brain Research, 691:25-36, (1995)).
This ex vivo therapy reduced Parkinson's-like symptoms in the rats up to 45% 32
days after transfer. Also, the ryrosine hydroxylase gene has been placed into astrocytes
with similar results (Lundberg et al., Develop. Neurol, 139:39-53 (1996) and references
However, such ex vivo systems have problems. In particular, refroviral vectors
currently used are down-regulated in vivo and the fransgene is only transiently expressed
(review by Mulligan, Science, 260:926-932 (1993)). Also, such studies used primary
cells, asfrocytes, which have finite life span and replicate slowly. Such properties
adversely affect the rate of transfection and impede selection of stably transfected cells. Moreover, it is almost impossible to propagate a large population of gene targeted
primary cells to be used in homologous recombination techniques.
By contrast, the difficulties associated with retroviral systems should be eliminated
by the use of human embryonic or stem-like cells. It has been demonstrated previously
by the subject assignee that cattle and pig embryonic cell lines can be transfected and
selected for stable integration of heterologous DNA. Such methods are described in
commonly assigned U.S. Serial No. 08/626,054, filed April 1, 1996, now U.S. Patent No.
5,905,042, incorporated by reference in its entirety. Therefore, using such methods or
other known methods, desired genes may be introduced into the subject human embryonic
or stem-like cells, and the cells differentiated into desired cell types, e.g., hematopoietic
cells, neural cells, pancreatic cells, cartilage cells, etc.
Genes which may be introduced into the subject embryonic or stem-like cells
include, by way of example, epidermal growth factor, basic fibroblast growth factor, glial
derived neurotrophic growth factor, insulin-like growth factor (I and II), neurotrophin-3,
neurotrophin-4/5, ciliary neurotrophic factor, AFT-1, cytokine genes (interleukins,
interferons, colony stimulating factors, tumor necrosis factors (alpha and beta), etc.),
genes encoding therapeutic enzymes, collagen, human serum albumin, etc.
In addition, it is also possible to use one of the negative selection systems now
known in the art for eliminating therapeutic cells from a patient if necessary. For
example, donor cells fransfected with the thymidine kinase (TK) gene will lead to the
production of embryonic cells containing the TK gene. Differentiation of these cells will lead to the isolation of therapeutic cells of interest which also express the TK gene. Such
cells may be selectively eliminated at any time from a patient upon gancyclovir
administration. Such a negative selection system is described in U.S. Patent No.
5,698,446, and is herein incorporated by reference.
The subject embryonic or stem-like cells, preferably human cells, also may be used
as an in vitro model of differentiation, in particular for the study of genes which are
involved in the regulation of early development.
Also, differentiated cell tissues and organs using the subject embryonic or stem¬
like cells may be used in drug studies.
Further, the subject cells may be used to express recombinant DNAs.
Still further, the subject embryonic or stem-like cells may be used as nuclear
donors for the production of other embryonic or stem-like cells and cell colonies.
Also, cultured inner cell mass, or stem cells, produced according to the invention
may be introduced into animals, e.g., SCID mice, cows, pigs, e.g., under the renal capsule
or intramuscularly and used to produce a teratoma therein. This teratoma can be used to
derive different tissue types. Also, the inner cell mass produced by X-species nuclear
transfer may be introduced together with a biodegradable, biocompatible polymer matrix
that provides for the formation of 3-dimensional tissues. After tissue formation, the
polymer degrades, ideally just leaving the donor tissue, e.g., cardiac, pancreatic, neural,
lung, liver. In some instances, it may be advantageous to include growth factors and
proteins that promote angiogenesis. Alternatively, the formation of tissues can be effected totally in vitro, with appropriate culture media and conditions, growth factors,
and biodegradable polymer matrices.
In order to more clearly describe the subject invention, the following examples are
MATERIALS AND METHODS
Donor Cells for Nuclear Transfer
Epithelial cells were lightly scraped from the inside of the mouth of a consenting
adult with a standard glass slide. The cells were washed off the slide into a petri dish
containing phosphate buffered saline without Ca or Mg. The cells were pipetted through
a small-bore pipette to break up cell clumps into a single cell suspension. The cells were
then transferred into a microdrop of TL-HEPES medium containing 10% fetal calf serum
(FCS) under oil for nuclear transfer into enucleated cattle oocytes.
Nuclear Transfer Procedures
Basic nuclear transfer procedures have been described previously. Briefly, after
slaughterhouse oocytes were matured in vitro the oocytes were stripped of cumulus cells
and enucleated with a beveled micropipette at approximately 18 hours post maturation
(hpm). Enucleation was confirmed in TL-HEPES medium plus bisbenzimide (Hoechst
33342, 3 μg/ml; Sigma). Individual donor cells were then placed into the perivitelline
space of the recipient oocyte. The bovine oocyte cytoplasm and the donor nucleus (NT
unit) are fused together using electrofusion techniques. One fusion pulse consisting of 90 V for 15 μsec was applied to the NT unit. This occurred at 24 hours post-initiation of
maturation (hpm) of the oocytes. The NT units were placed in CRlaa medium until 28
The procedure used to artificially activate oocytes has been described elsewhere.
NT unit activation was at 28 hpm. A brief description of the activation procedure is as
follows: NT units were exposed for four min to ionomycin (5 μM; CalBiochem, La Jolla,
CA) in TL-HEPES supplemented with 1 mg/ml BSA and then washed for five rnin in TL-
HEPES supplemented with 30 mg/ml BSA. The NT units were then transferred into a
microdrop of CRlaa culture medium containing 0.2 mM DMAP (Sigma) and cultured at
38.5 °C 5% C02 for four to five hours. The NT units were washed and then placed in a
CRlaa medium plus 10% FCS and 6 mg/ml BSA in four well plates containing a
confluent feeder layer of mouse embryonic fibroblasts (described below). The NT units
were cultured for three more days at 38.5° C and 5% C02. The culture medium was
changed every three days until day 12 after the time of activation. At this time NT units
reaching the desired cell number, i.e., about 50 cell number, were mechanically removed
from the zona and used to produce embryonic cell lines. A photograph of an NT unit
obtained as described above is contained in Figure 1.
Fibroblast feeder layer
Primary cultures of embryonic fibroblasts were obtained from 14-16 day old
murine fetuses. After the head, liver, heart and alimentary tract were aseptically removed,
the embryos were minced and incubated for 30 minutes at 37 °C in pre- warmed trypsin EDTA solution (0.05% trypsin/0.02% EDTA; GIBCO, Grand Island, NY). Fibroblast
cells were plated in tissue culture flasks and cultured in alpha-MEM medium
(BioWhittaker, Walkersville, MD) supplemented with 10% fetal calf serum (FCS)
(Hyclone, Logen, UT), penicillin (100 IU/ml) and streptomycin (50 μl/ml). Three to four
days after passage, embryonic fibroblasts, in 35 x 10 Nunc culture dishes (Baxter
Scientific, McGaw Park, EL), were irradiated. The irradiated fibroblasts were grown and
maintained in a humidified atmosphere with 5% C02 in air at 37°C. The culture plates
which had a uniform monolayer of cells were then used to culture embryonic cell lines.
Production of embryonic cell line.
NT unit cells obtained as described above were washed and plated directly onto
irradiated feeder fibroblast cells. These cells included those of the inner portion of the
NT unit. The cells were maintained in a growth medium consisting of alpha MEM
supplemented with 10% FCS and 0.1 mM beta-mercaptoethanol (Sigma). Growth
medium was exchanged every two to three days. The initial colony was observed by the
second day of culture. The colony was propagated and exhibits a similar morphology to
previously disclosed mouse embryonic stem (ES) cells. Individual cells within the colony
are not well defined and the perimeter of the colony is refractile and smooth in ap¬
pearance. The cell colony appears to have a slower cell doubling time than mouse ES
cells. Also, unlike bovine and porcine derived ES cells, the colony does not have an
epithelial appearance thus far. Figures 2 through 5 are photographs of ES-like cell
colonies obtained as described, supra. Production of Differentiated Human Cells
The human embryonic cells obtained are transferred to a differentiation medium
and cultured until differentiated human cell types are obtained.
Table 1. Human cells as donor nuclei in NT unit production and development.
The one NT unit that developed a structure having greater than 16 cells was plated
down onto a fibroblast feeder layer. This structure was attached to the feeder layer and
started to propagate forming a colony with a ES cell-like morphology (See, e.g., Figure
2). Moreover, although the 4 to 16 cell stage structures were not used to try and produce
an ES cell colony, it has been previously shown that this stage is capable of producing ES
or ES-like cell lines (mouse, Eistetter et al., Devel Growth and Differ., 31:275-282
(1989); Bovine, Stice et al., 1996)). Therefore, it is expected that 4 - 16 cell stage NT
units should also give rise to embryonic or stem-like cells and cell colonies. Also, similar results were obtained upon fusion of an adult human keratinocyte cell
line with an enucleated bovine oocyte, which was cultured in media comprising ACM,
uridine, glucose, and 1000 IU of LLF. Out of 50 reconstructed embryos, 22 cleaved and
one developed into a blastocyst at about day 12. This blastocyst was plated and the
production of an ES cell line is ongoing.
A. Mitochondria Isolation Protocol from a Cell
This Example relates to isolation of mitochondria and use thereof to enhance the
efficiency of cross-species nuclear transfer. The number of mitochondria per cell varies
from cell line to cell line. For example, mouse L cells contain - 100 mitochondria per
cell, whereas there are at least twice that number in HeLa cells. The cells are swollen in
a hypotonic buffer and ruptured with a few strokes in a Dounce homogenizer using a
tight-fitting pestle, and the mitochondria are isolated by differential centrifugation.
The solutions, tubes, and homogenizer should be pre-chilled on ice. All
centrifugation steps are at 40 °C. This protocol is based on starting with a washed cell
pellet of 1-2 ml. The cell pellet is resuspended in 11 ml of ice-cold RSB and transferred
to a 16 ml Dounce homogenizer.
RSB (A hypotonic buffer for swelling the tissue culture cells)
lO mM NaCl
1.5 mM MgCl2 10 mM Tris-HCl, pH 7.5
The cells are allowed to swell for five to ten minutes. The progress of the swelling
is maintained using a phase contrast microscope. The swollen cells are replaced,
preferably by several strokes with a pestle. Immediately after, 8 ml of 2.5x MS buffer are
added to give a final concentration of lx MS. The top of the homogenizer is then covered
with Parafilm and mixed by inverting a couple of times.
2.5x MS Buffer
525 mM mannitol
175 Mm sucrose
215 mM EDTA pH 7.5
lx MS Buffer
210 mM mannitol
70 mM Sucrose
5 mM Tris-HCl, pH 7.5
1 mM EDTA, pH 7.5
MS Buffer is an iso-osmotic buffer to maintain the tonicity of the
organelles and prevent agglutination.
Thereafter, the homogenate is transferred to a centrifuge tube for differential
centrifugation. The homogenizer is rinsed with a small amount of MS buffer and added to the homogenate. The volume is brought to 30 ml with MS buffer. The homogenate
is then centrifuged at 1300 g for five minutes to remove nuclei, unbroken cells, and large
membrane fragments. The supernatant is then poured into a clean centrifuge tube. The
nuclear spin-down is repeated twice. The supernatant is then transferred to a clean
centrifuge tube and a pellet containing the mitochondria is centrifuged at 17,000 g for 15
minutes. The supernatant is discarded and the inside of the tube wiped with a Kimwipe.
The mitochondria is washed by re-suspending the pellet in IX MS and repeating the
17,000 g sedimentation. The supernatant is discarded and the pellet is resuspended in a
buffer. Mitochondria can be stored at -80 °C for prolonged periods, e.g., up to a year, but
preferably will be used shortly thereafter for NT.
This basic protocol can be modified. In particular, it may be desirable to further
isolate mitochondrial DNA and us same for NT. In such case, contamination with nuclei,
not small organelles, potentially is a problem and the following modifications may be
made. For example, the cells may be harvested in stationary growth phase when the
fewest cells are actively dividing, and CaCl2 substituted for MgCl2 in the RSB to stabilize
the nuclear membrane. The washing of the mitochondrial pellet is omitted as is the
density gradient purification. Instead, the mitochondrial pellet is simply resuspended and
lysed, and the mitochondrial DNA purified from any remaining nuclear DNA. As noted,
suitable methods for purifying mitachondria and mitochondrial DNA are well known in
the art. Homogenization works best if the cells are resuspended in at least 5-1 OX the
volume of the cell pellet and if the cell suspension fills the homogenizer at least half full.
Press the homogenizer pestle straight down the tube, maintaining a firm, steady pressure.
The Dounce homogenizer disrupts swollen tissue culture cells by pressure change. As
the pestle is pressed down, pressure around the cell increases; when the cell slips past the
end of the pestle, the sudden decrease in pressure causes the cell to rupture. If the pestle
is very tight fitting, there may be some mechanical breakage as well.
B. Isolation of Mitochondria From Tissue
A mitochondrial isolation protocol is selected based on the particular tissue. For
example, the homogenization buffer should be optimized for the tissue, and the optimal
way to homogenize the tissue utilized. Suitable methods are well known in the art.
Rat liver is the most frequently used tissue for mitochondrial preparations because
it is readily available, is easy to homogenize, and the cells contain a large number of
mitochondria ( 1000-2000 per cell). For example, a motor-driven, Teflon and glass Potter-
Elvehjem homogenizer can be used homogenize rat liver. Alternatively, if the tissue is
soft enough, a Dounce homogenizer with a loose pestle can be used. The yield and purity
of the mitochondrial preparation is influenced by the method of preparation, speed of
preparation, and the age and physiological condition of the animal. As noted, methods
of purifying mitochondria are well known. Preferably, the buffer, tubes, and homogenizer will be pre-chilled. Pre-chilling a
glass and Teflon type homogenizer creates the proper gap between the tube and pestle.
The centrifugation steps are preferably effected at 40 °C.
Essentially, the process will comprise removal of the liver, taking care not to
rupture the gall bladder. This is placed in a beaker on ice and any connective tissue is
removed. The tissue is recognized and returned to the beaker, e.g., using very sharp
scissors, a scalpel, or razor blade, mince it into 1-2 slices. The pieces are then rinsed,
preferably twice, with homogenization buffer (IX MS) to remove most of the blood, and
the tissue transferred to the homogenizer tube. Enough homogenization buffer if added
to prepare a 1 : 10 (w/v) homogenate.
Use of Isolated Mitochondria or Mitochondrial DNA to Enhance NT Efficacy
It is theorized by the inventors that the efficacy of cross-species nuclear transfer
may be enhanced by introduction of mitochondria or mitochondrial DNA at the same
species as donor cell or nucleus. Thereupon, the nucleus DNA of resultant NT units will
be species compatible.
Mitochondria isolated by the above or other known procedures are incorporated,
typically by injection, into any of the following (in the case of human donor cell/bovine
oocyte nuclear transfer):
(i) non-activated, non-enucleated bovine oocytes;
(ii) non-activated, enucleated bovine oocytes;
(iii) activated, enucleated bovine oocytes; (iv) non-activated, fused (with human donor cell or nucleus"! bovine nnr.vtes-
(v) activated, fused and cleaved reconstructed (cow oocyte/human cell)
(vi) activated, fused one cell reconstructed (cow oocyte/human cell) embryo.
The same procedures will enhance other cross-species NTs. Essentially,
mitochondria will again be introduced into any of (i)-(vi) of the same species as the donor
cell or nucleus, and the oocyte will be of a different species origin. Generally about 1 to
200 picoliters of mitochondrial suspension are injected into any of the above. The
introduction of such mitochondria will result in NT units wherein the mitochondrial and
donor DNA are compatible.
Another method for improving the efficacy of the cross-species nuclear transfer
comprises the fusion of one or more enucleated somatic cells, typically human (of the
same species as donor cell or nucleus), with any of the following:
(i) non-activated, non-enucleated (e.g., bovine) oocyte;
(ii) non-activated, enucleated (e.g., bovine) oocyte;
(iii) activated, enucleated (e.g., bovine) oocyte;
(iv) non-activated, fused (with human cell) oocyte (typically bovine);
(v) activated, fused and cleaved reconstructed (e.g., cow oocyte/human cell)
embryo; (vi) activated, fused one cell reconstructed (cow oocyte/human cell) embryo;
(vii) non-activated, fused (e.g., with human cell) oocyte (typically bovine
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.
Methods for the large-scale enucleation of cells with cytochalasin B are well
known in the art. Enucleation is preferably effected using the monolayer technique. This
method uses small numbers of cells attached to the growth surface of a culture disc and
is ideal if limited numbers of donor cells are available. Another suitable procedure, the
gradient technique, requires centrifugation of cells through Ficoll gradients and is best
suited for enucleation of large number (>107) of cells.
Monolayer Technique. The monolayer technique is ideal for virtually any cell
which grows attached to the growth surface.
Polycarbonate or polypropylene 250-ml wide-mouth centrifuge bottles with screw-
top caps are sterilized by autoclaving. The caps preferably are autoclaved separately from
the bottle to prevent damage to the centrifuge bottle. The bottle are prepared for the
enucleation procedure by the sterile addition of 30 ml DMEM, 2 ml bovine serum, and 0.32 ml cytochalasin B (1 mg/ml) to each. The caps are placed on the bottles, and the
bottles are maintained at 37° prior to use.
The cells to be enucleated (from a few hundred to ~ 105 cells) are seeded on a
culture dish (35 x 15 mm; Nunc Inc., Naperville, IL). Typically, the cells are grown for
at least twenty-four hours on the dishes to promote maximal attachment to the growth
surface. Preferably, the cells are prevented from becoming confluent. The culture dish
is prepared for centrifugation by wiping the outside of the bottom half of the dish
(containing the cells) with 70% (v/v) ethanol for the purpose of sterilization.
Alternatively, the dish can be kept sterile during cell culturing by maintaining it within
a larger, sterile culture dish. The medium is removed from the dish and the dish (without
top) is placed upside down within the centrifuge bottle.
The rotor (GSA, DuPont, Wilmington, DE) and centrifuge are preferably pre-
warmed to 37° by centrifugation for 30-45 minutes at 8000 rpm. The HS-4 swinging-
bucket rotor (DuPont) can alternatively be used. The optimal time and speed of
centrifugation varies for each cell type. For myoblasts and fibroblasts, the centrifuge
bottle with the culture dish is placed in the pre-warmed rotor and centrifuged for
approximately 20 minutes (interval between the time when the rotor reaches the desired
speed and the time when the centrifuge is turned off). Preferably, speeds of 6500 to 7200
rpm are used.
After centrifugation, the centrifuge bottle is removed from the rotor, and the
culture plate is removed from the bottles with forceps. A small amount of medium is maintained in the plate to keep the cells moist in order to maintain cell viability. The
outside of the dish, including the top edge, is wiped with a sterile wiper, then moistened
with 95% (v/v) ethanol, to remove any medium and to dry it. A sterile top is placed onto
the dish. If the enucleated cells are not going to be used immediately, complete culture
medium (medium supplemented with the appropriate concentration of serum) should be
added to the dish, and the cells placed in a C02 incubator. The resultant enucleated cells
(karyoplast) are fused with any of (i) - (viii) above.
While the present invention has been described and illustrated herein by reference
to various specific materials, procedures, and examples, it is understood that the invention
is not restricted to the particular material, combinations of materials, and procedures
selected for that purpose. Numerous variations of such details can be implied and will
be appreciated by those skilled in the art.
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|WO1998007841A1 *||28 Jul 1997||26 Feb 1998||University Of Massachusetts||Embryonic or stem-like cell lines produced by cross species nuclear transplantation|
|WO1999045100A1 *||2 Mar 1999||10 Sep 1999||University Of Massachusetts, A Public Institution Of Higher Education Of The Commonwealth Of Massachusetts, As Represented By Its Amherst Campus||Embryonic or stem-like cell lines produced by cross-species nuclear transplantation|
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|4||*||THOMSON J.A. ET AL.: "Embryonic stem cell lines derived from human blastocysts", SCIENCE, vol. 282, no. 6, 6 November 1998 (1998-11-06), pages 1145 - 1147, XP002933311|
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|Patente citante||Fecha de presentación||Fecha de publicación||Solicitante||Título|
|EP1214403A1 *||6 Sep 2000||19 Jun 2002||Advanced Cell Technology, Inc.||Method for generating immune-compatible cells and tissues using nuclear transfer techniques|
|EP1214403A4 *||6 Sep 2000||9 Mar 2005||Advanced Cell Tech Inc||Method for generating immune-compatible cells and tissues using nuclear transfer techniques|
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|EP1226239A4 *||13 Oct 2000||12 Feb 2003||Advanced Cell Tech Inc||Methods of producing differentiated progenitor cells and lineage-defective embryonic stem cells|
|EP1240318A1 *||10 May 2000||18 Sep 2002||University of Massachusetts, a Public Institution of Higher Education of The Commonwealth of Massachusetts,||Embryonic or stem-like cells produced by cross species nuclear transplantation|
|EP1240318A4 *||10 May 2000||8 Dic 2004||Univ Massachusetts||Embryonic or stem-like cells produced by cross species nuclear transplantation|
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|Clasificación internacional||A61K35/36, A61P25/16, A61K35/407, A61K35/32, A61P3/10, A61P25/00, A61K35/34, A61K35/48, A61K35/28, A61P35/00, A61P7/00, A61P1/16, A61P31/18, A61P9/00, A61K35/38, A61P25/14, C12N5/10, A61P13/02, A61K35/30, A61P21/04, C12N5/02, A61K35/12, A61K35/22, C12N15/09, A61K35/39, A61P43/00, A61P17/02, A61P25/28, A61K48/00, C12N15/873|
|Clasificación cooperativa||A61K35/12, C12N2517/04, C12N15/873|
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