U.S. patent application number 09/761289 was filed with the patent office on 2001-09-27 for primate embryonic stem cells.
Invention is credited to Thomson, James A..
Application Number | 20010024825 09/761289 |
Document ID | / |
Family ID | 23484566 |
Filed Date | 2001-09-27 |
United States Patent
Application |
20010024825 |
Kind Code |
A1 |
Thomson, James A. |
September 27, 2001 |
Primate embryonic stem cells
Abstract
A purified preparation of primate embryonic stem cells is
disclosed. This preparation is characterized by the following cell
surface markers: SSEA-1 (-); SSEA-4 (+); TRA-1-60 (+); TRA-1-81
(+); and alkaline phosphatase (+). In a particularly advantageous
embodiment, the cells of the preparation are human embryonic stem
cells, have normal karyotypes, and continue to proliferate in an
undifferentiated state after continuous culture for eleven months.
The embryonic stem cell lines also retain the ability, throughout
the culture, to form trophoblast and to differentiate into all
tissues derived from all three embryonic germ layers (endoderm,
mesoderm and ectoderm). A method for isolating a primate embryonic
stem cell line is also disclosed.
Inventors: |
Thomson, James A.; (Madison,
WI) |
Correspondence
Address: |
Nicholas J. Seay
Quarles & Brady LLP
1 South Pinckney Street
P O Box 2113
Madison
WI
53701-2113
US
|
Family ID: |
23484566 |
Appl. No.: |
09/761289 |
Filed: |
January 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09761289 |
Jan 16, 2001 |
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09106390 |
Jun 26, 1998 |
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6200806 |
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09106390 |
Jun 26, 1998 |
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08591246 |
Jan 18, 1996 |
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5843780 |
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09106390 |
Jun 26, 1998 |
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08376327 |
Jan 20, 1995 |
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Current U.S.
Class: |
435/366 |
Current CPC
Class: |
C12N 5/0606 20130101;
C12N 5/0603 20130101; C12N 2506/02 20130101; C12N 2502/13 20130101;
A61P 37/00 20180101; C12N 5/0605 20130101; A61P 3/10 20180101; A61P
25/16 20180101 |
Class at
Publication: |
435/366 |
International
Class: |
C12N 005/08 |
Goverment Interests
[0002] This invention was made with United States government
support awarded by NIH NCRR Grant No. RR00167. The United States
government has certain rights in this invention.
Claims
I claim:
1. A purified preparation of human embryonic stem cells which (i)
is capable of proliferation in an in vitro culture for over one
year, (ii) maintains a karyotype in which all the chromosomes
characteristic of the human species are present and not noticeably
altered through prolonged culture, (iii) maintains the potential to
differentiate to derivatives of endoderm, mesoderm, and ectoderm
tissues throughout the culture, and (iv) are inhibited from
differentiation when cultured on a fibroblast feeder layer.
2. The preparation of claim 1, wherein the stem cells will
spontaneously differentiate to trophoblast and produce chorionic
gonadotropin when cultured to high density.
3. A purified preparation of human embryonic stem cells wherein the
cells are essentially negative for the SSEA-1 marker, positive for
the SSEA-4 marker, express alkaline phosphatase activity, are
pluripotent, and have karyotypes which includes the presence of all
of the chromosomes characteristic of the human species and in which
none of the chromosomes are noticeably altered.
4. The preparation of claim 3, wherein the cells are positive for
the TRA-1-60, and TRA-1-81 markers.
5. The preparation of claim 3, wherein the cells continue to
proliferate in an undifferentiated state after continuous culture
for at least one year.
6. The preparation of claim 3, wherein the cells will differentiate
to trophoblast when cultured beyond confluence and will produce
chorionic gonadotropin.
7. The preparation of claim 3, wherein the cells remain euploid for
more than one year of continuous culture.
8. The preparation of claim 3, wherein the cells differentiate into
cells derived from mesoderm, endoderm and ectoderm germ layers when
the cells are injected into a SCID mouse.
9. A method of isolating a human embryonic stem cell line,
comprising the steps of: (a) isolating a human blastocyst; (b)
isolating cells from the inner cell mass of the blastocyte of (a);
(c) plating the inner cell mass cells on embryonic fibroblasts,
wherein inner cell mass-derived cell masses are formed; (d)
dissociating the mass into dissociated cells; (e) replating the
dissociated cells on embryonic feeder cells; (f) selecting colonies
with compact morphologies and cells with high nucleus to cytoplasm
ratios and prominent nucleoli; and (g) culturing the cells of the
selected colonies.
10. A method as claimed in claim 9, further comprising maintaining
the isolated cells on a fibroblast feeder layer to prevent
differentiation.
11. A cell line developed by the method of step 9.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. Ser. No. 08/591,246
which was filed on Jan. 18, 1996, and is a continuation-in-part of
U.S. Ser. No. 08/376,327 which was filed on Jan. 20, 1995.
BACKGROUND OF THE INVENTION
[0003] In general, the field of the present invention is stem cell
cultures. Specifically, the field of the present invention is
primate embryonic stem cell cultures.
[0004] In general, stem cells are undifferentiated cells which can
give rise to a succession of mature functional cells. For example,
a hematopoietic stem cell may give rise to any of the different
types of terminally differentiated blood cells. Embryonic stem (ES)
cells are derived from the embryo and are pluripotent, thus
possessing the capability of developing into any organ or tissue
type or, at least potentially, into a complete embryo.
[0005] One of the seminal achievements of mammalian embryology of
the last decade is the routine insertion of specific genes into the
mouse genome through the use of mouse ES cells. This alteration has
created a bridge between the in vitro manipulations of molecular
biology and an understanding of gene function in the intact animal.
Mouse ES cells are undifferentiated, pluripotent cells derived in
vitro from preimplantation embryos (Evans, et al. Nature
292:154-159, 1981; Martin, Proc. Natl. Acad. Sci. USA 78:7634-7638,
1981) or from fetal germ cells (Matsui, et al., Cell 70:841-847,
1992). Mouse ES cells maintain an undifferentiated state through
serial passages when cultured in the presence of fibroblast feeder
layers in the presence of Leukemia Inhibitory Factor (LIF)
(Williams, et al., Nature 336:684-687, 1988). If LIF is removed,
mouse ES cells differentiate.
[0006] Mouse ES cells cultured in non-attaching conditions
aggregate and differentiate into simple embryoid bodies, with an
outer layer of endoderm and an inner core of primitive ectoderm. If
these embryoid bodies are then allowed to attach onto a tissue
culture surface, disorganized differentiation occurs of various
cell types, including nerves, blood cells, muscle, and cartilage
(Martin, 1981, supra; Doetschman, et al., J. Embryol. Exp. Morph.
87:27-45, 1985). Mouse ES cells injected into syngeneic mice form
teratocarcinomas that exhibit disorganized differentiation, often
with representatives of all three embryonic germ layers. Mouse ES
cells combined into chimeras with normal preimplantation embryos
and returned to the uterus participate in normal development
(Richard, et al., Cytogenet. Cell Genet. 65:169-171, 1994).
[0007] The ability of mouse ES cells to contribute to functional
germ cells in chimeras provides a method for introducing
site-specific mutations into mouse lines. With appropriate
transfection and selection strategies, homologous recombination can
be used to derive ES cell lines with planned alterations of
specific genes. These genetically altered cells can be used to form
chimeras with normal embryos and chimeric animals are recovered. If
the ES cells contribute to the germ line in the chimeric animal,
then in the next generation a mouse line for the planned mutation
is established.
[0008] Because mouse ES cells have the potential to differentiate
into any cell type in the body, mouse ES cells allow the in vitro
study of the mechanisms controlling the differentiation of specific
cells or tissues. Although the study of mouse ES cells provides
clues to understanding the differentiation of general mammalian
tissues, dramatic differences in primate and mouse development of
specific lineages limits the usefulness of mouse ES cells as a
model of human development. Mouse and primate embryos differ
meaningfully in the timing of expression of the embryonic genome,
in the formation of an egg cylinder versus an embryonic disc
(Kaufman, The Atlas of Mouse Development, London: Academic Press,
1992), in the proposed derivation of some early lineages (O'Rahilly
& Muller, Developmental Stages in Human Embryos, Washington:
Carnegie Institution of Washington, 1987), and in the structure and
function in the extraembryonic membranes and placenta (Mossman,
Vertebrate Fetal Membranes, New Brunswick: Rutgers, 1987). Other
tissues differ in growth factor requirements for development (e.g.
the hematopoietic system (Lapidot et al., Lab An Sci 43:147-149,
1994)), and in adult structure and function (e.g. the central
nervous system). Because humans are primates, and development is
remarkably similar among primates, primate ES cells lines will
provide a faithful model for understanding the differentiation of
primate tissues in general and human tissues in particular.
[0009] The placenta provides just one example of how primate ES
cells will provide an accurate model of human development that
cannot be provided by ES cells from other species. The placenta and
extraembryonic membranes differ dramatically between mice and
humans. Structurally, the mouse placenta is classified as
labyrinthine, whereas the human and the rhesus monkey placenta are
classified as villous. Chorionic gonadotropin, expressed by the
trophoblast, is an essential molecule involved in maternal
recognition of pregnancy in all primates, including humans (Hearn,
J Reprod Fertil 76:809-819, 1986; Hearn et al., J Reprod Fert
92:497-509, 1991). Trophoblast secretion of chorionic gonadotropin
in primates maintains the corpus luteum of pregnancy and, thus,
progesterone secretion. Without progesterone, pregnancy fails. Yet
mouse trophoblast produces no chorionic gonadotropin, and mice use
entirely different mechanisms for pregnancy maintenance (Hearn et
al., "Normal and abnormal embryo-fetal development in mammals," In:
Lamming E, ed. Marshall's Physiology of Reproduction. 4th ed.
Edinburgh, N.Y.: Churchill Livingstone, 535-676, 1994). An
immortal, euploid, primate ES cell line with the developmental
potential to form trophoblast in vitro, will allow the study of the
ontogeny and function of genes such as chorionic gonadotropin which
are critically important in human pregnancy. Indeed, the
differentiation of any tissue for which there are significant
differences between mice and primates will be more accurately
reflected in vitro by primate ES cells than by mouse ES cells.
[0010] The major in vitro models for studying trophoblast function
include human choriocarcinoma cells, which are malignant cells that
may not faithfully reflect normal trophectoderm; short-term primary
cultures of human and non-human primate cytotrophoblast, which in
present culture conditions quickly form non-dividing syncytial
trophoblast; and in vitro culture of preimplantation non-human
primate embryos (Hearn, et al., J. Endocrinol. 119:249-255, 1988;
Coutifaris, et al., Ann. NY Acad. Sci. 191-201, 1994). An immortal,
euploid, non-human primate embryonic stem (ES) cell line with the
developmental potential to form trophectoderm offers significant
advantages over present in vitro models of human trophectoderm
development and function, as trophoblast-specific genes such as
chorionic gonadotropin could be stably altered in the ES cells and
then studied during differentiation to trophectoderm.
[0011] The cell lines currently available that resembles primate ES
cells most closely are human embryonic carcinoma (EC) cells, which
are pluripotent, immortal cells derived from teratocarcinomas
(Andrews, et al., Lab. Invest. 50(2):147-162, 1984; Andrews, et
al., in: Robertson E., ed. Teratocarcinomas and Embryonic Stem
Cells: A Practical Approach. Oxford: IRL press, pp. 207-246, 1987).
EC cells can be induced to differentiate in culture, and the
differentiation is characterized by the loss of specific cell
surface markers (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) and the
appearance of new markers (Andrews, et al., 1987, supra). Human EC
cells will form teratocarcinomas with derivatives of multiple
embryonic lineages in tumors in nude mice. However, the range of
differentiation of these human EC cells is limited compared to the
range of differentiation obtained with mouse ES cells, and all EC
cell lines derived to date are aneuploid (Andrews, et al., 1987,
supra). Similar mouse EC cell lines have been derived from
teratocarcinomas, and, in general their developmental potential is
much more limited than mouse ES cells (Rossant, et al., Cell
Differ. 15:155-161, 1984). Teratocarcinomas are tumors derived from
germ cells, and although germ cells (like ES cells) are
theoretically totipotent (i.e. capable of forming all cell types in
the body), the more limited developmental potential and the
abnormal karyotypes of EC cells are thought to result from
selective pressures in the teratocarcinoma tumor environment
(Rossant & Papaioannou, Cell Differ 15:155-161, 1984). ES
cells, on the other hand, are thought to retain greater
developmental potential because they are derived from normal
embryonic cells in vitro, without the selective pressures of the
teratocarcinoma environment. Nonetheless, mouse EC cells and mouse
ES cells share the same unique combination of cell surface markers
(SSEA-1 (+), SSEA-3 (-), SSEA-4 (-), and alkaline phosphatase
(+)).
[0012] Pluripotent cell lines have also been derived from
preimplantation embryos of several domestic and laboratory animals
species (Evans, et al., Theriogenology 33(1):125-128, 1990; Evans,
et al., Theriogenology 33(1):125-128, 1990; Notarianni, et al., J.
Reprod. Fertil. 41(Suppl.):51-56, 1990; Giles, et al., Mol. Reprod.
Dev. 36:130-138, 1993; Graves, et al., Mol. Reprod. Dev.
36:424-433, 1993; Sukoyan, et al., Mol. Reprod. Dev. 33:418-431,
1992; Sukoyan, et al., Mol. Reprod. Dev. 36:148-158, 1993;
Iannaccone, et al., Dev. Biol. 163:288-292, 1994).
[0013] Whether or not these cell lines are true ES cells lines is a
subject about which there may be some difference of opinion. True
ES cells should: (i) be capable of indefinite proliferation in
vitro in an undifferentiated state; (ii) maintain a normal
karyotype through prolonged culture; and (iii) maintain the
potential to differentiate to derivatives of all three embryonic
germ layers (endoderm, mesoderm, and ectoderm) even after prolonged
culture. Strong evidence of these required properties have been
published only for rodents ES cells including mouse (Evans &
Kaufman, Nature 292:154-156, 1981; Martin, Proc Natl Acad Sci USA
78:7634-7638, 1981) hamster (Doetschmanet al. Dev Biol 127:224-227,
1988), and rat (Iannaccone et al. Dev Biol 163:288-292, 1994), and
less conclusively for rabbit ES cells (Gileset al. Mol Reprod Dev
36:130-138, 1993; Graves & Moreadith, Mol Reprod Dev
36:424-433, 1993). However, only established ES cell lines from the
rat (Iannaccone, et al., 1994, supra) and the mouse (Bradley, et
al., Nature 309:255-256, 1984) have been reported to participate in
normal development in chimeras. There are no reports of the
derivation of any primate ES cell line.
BRIEF SUMMARY OF THE INVENTION
[0014] The present invention is a purified preparation of primate
embryonic stem cells. The primate ES cell lines are true ES cell
lines in that they: (i) are capable of indefinite proliferation in
vitro in an undifferentiated state; (ii) are capable of
differentiation to derivatives of all three embryonic germ layers
(endoderm, mesoderm, and ectoderm) even after prolonged culture;
and (iii) maintain a normal karyotype throughout prolonged culture.
The true primate ES cells lines are therefore pluripotent.
[0015] The present invention is also summarized in that primate ES
cell lines are preferably negative for the SSEA-1 marker,
preferably positive for the SSEA-3 marker, and positive for the
SSEA-4 marker. The primate ES cell lines are also positive for the
TRA-1-60, and TRA-1-81 markers, as well as positive for the
alkaline phosphatase marker.
[0016] It is an advantageous feature of the present invention that
the primate ES cell lines continue to proliferate in an
undifferentiated state after continuous culture for at least one
year. In a particularly advantageous embodiment, the cells remain
euploid after proliferation in an undifferentiated state.
[0017] It is a feature of the primate ES cell lines in accordance
with the present invention that the cells can differentiate to
trophoblast in vitro and express chorionic gonadotropin.
[0018] The present invention is also a purified preparation of
primate embryonic stem cells that has the ability to differentiate
into cells derived from mesoderm, endoderm, and ectoderm germ
layers after the cells have been injected into an immunocompromised
mouse, such as a SCID mouse.
[0019] The present invention is also a method of isolating a
primate embryonic stem cell line. The method comprises the steps of
isolating a primate blastocyst, isolating cells from the inner
cellular mass (ICM) of the blastocyst, plating the ICM cells on a
fibroblast layer (wherein ICM-derived cell masses are formed)
removing an ICM-derived cell mass and dissociating the mass into
dissociated cells, replating the dissociated cells on embryonic
feeder cells and selecting colonies with compact morphology
containing cells with a high nucleus/cytoplasm ratio, and prominent
nucleoli. The cells of the selected colonies are then cultured.
[0020] It is an object of the present invention to provide a
primate embryonic stem cell line.
[0021] It is an object of the present invention to provide a
primate embryonic stem cell line characterized by the following
markers: alkaline phosphatase(+); SSEA-1(-); preferably SSEA-3(+);
SSEA-4(+); TRA-1-60(+); and TRA-1-81(+).
[0022] It is an object of the present invention to provide a
primate embryonic stem cell line capable of proliferation in an
undifferentiated state after continuous culture for at least one
year. Preferably, these cells remain euploid.
[0023] It is another object of the present invention to provide a
primate embryonic stem cell line wherein the cells differentiate
into cells derived from mesoderm, endoderm, and ectoderm germ
layers when the cells are injected into an immunocompromised
mouse.
[0024] Other objects, features, and advantages of the present
invention will become obvious after study of the specification,
drawings, and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a photomicrograph illustrating normal XY karyotype
of rhesus ES cell line R278.5 after 11 months of continuous
culture.
[0026] FIGS. 2A-2D are a set of phase-contrast photomicrographs
demonstrating the morphology of undifferentiated rhesus ES (R278.5)
cells and of cells differentiated from R278.5 in vitro
(bar=100.mu.). Photograph 2A demonstrates the distinct cell
borders, high nucleus to cytoplasm ratio, and prominent nucleoli of
undifferentiated rhesus ES cells. Photographs 2B-2D shows
differentiated cells eight days after plating R278.5 cells on gel
treated tissue culture plastic (with 10.sup.3 units/ml added human
LIF). Cells of these three distinct morphologies are consistently
present when R278.5 cells are allowed to differentiate at low
density without fibroblasts either in the presence or absence of
soluble human LIF.
[0027] FIGS. 3A-F are photomicrographs demonstrating the expression
of cell surface markers on undifferentiated rhesus ES (R278.5)
cells (bar=100.mu.). Photograph 3A shows Alkaline Phosphatase (+);
Photograph 3B shows SSEA-1 (-); Photograph 3C shows SSEA-3 (+);
Photograph 3D shows SSEA-4 (+); Photograph 3E shows TRA-1-60 (+);
and Photograph 3F shows TRA-1-81 (+).
[0028] FIGS. 4A-4B are photographs illustrating expression of
.alpha.-fetoprotein mRNA and .alpha.- and .beta.-chorionic
gonadotrophin mRNA expression in rhesus ES cells (R278.5) allowed
to differentiate in culture.
[0029] FIGS. 5A-5F include six photomicrographs of sections of
tumors formed by injection of 0.5.times.10.sup.6 rhesus ES (R278.5)
cells into the hindleg muscles of SCID mice and analyzed 15 weeks
later. Photograph 5A shows a low power field demonstrating
disorganized differentiation of multiple cell types. A gut-like
structure is encircled by smooth muscle(s), and elsewhere foci of
cartilage (c) are present (bar=400.mu.); Photograph 5B shows
striated muscle (bar=40.mu.); Photograph 5C shows stratified
squamous epithelium with several hair follicles. The labeled hair
follicle (f) has a visible hair shaft (bar=200.mu.); Photograph 5D
shows stratified layers of neural cells in the pattern of a
developing neural tube. An upper "ventricular" layer, containing
numerous mitotic figures (arrows), overlies a lower "mantle" layer.
(bar=100.mu.); Photograph 5E shows ciliated columnar epithelium
(bar=40.mu.); Photograph 5F shows villi covered with columnar
epithelium with interspersed mucus-secreting goblet cells
(bar=200.mu.).
[0030] FIGS. 6A-6B include photographs of an embryoid Body. This
embryoid body was formed from a marmoset ES cell line (Cj62) that
had been continuously passaged in vitro for over 6 months.
Photograph 6A (above) shows a section of the anterior 1/3 of the
embryonic disc. Note the primitive ectoderm (E) forms a distinct
cell layer from the underlying primitive endoderm (e), with no
mixing of the cell layers. Note also that amnion (a) is composed of
two distinct layers; the inner layer is continuous with the
primitive ectoderm at the margins. Photograph 6B (below) shows a
section in the caudal 1/3 of embryonic disc. Note central groove
(arrow) and mixing of primitive ectoderm and endoderm representing
early primitive streak formation, indicating the beginning of
gastrulation. 400X, toluidine blue stain.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0031] (1) In General
[0032] (a) Uses of Primate ES Cells
[0033] The present invention is a pluripotent, immortal euploid
primate ES cell line, as exemplified by the isolation of ES cell
lines from two primate species, the common marmoset (Callithrix
jacchus) and the rhesus monkey (Macaca mulatta). Primate embryonic
stem cells are useful for:
[0034] (i) Generating transgenic non-human primates for models of
specific human genetic diseases. Primate embryonic stem cells will
allow the generation of primate tissue or animal models for any
human genetic disease for which the responsible gene has been
cloned. The human genome project will identify an increasing number
of genes related to human disease, but will not always provide
insights into gene function. Transgenic nonhuman primates will be
essential for elucidating mechanisms of disease and for testing new
therapies.
[0035] (ii) Tissue transplantation. By manipulating culture
conditions, primate ES cells, human and nonhuman, can be induced to
differentiate to specific cell types, such as blood cells, neuron
cells, or muscle cells. Alternatively, primate ES cells can be
allowed to differentiate in tumors in SCID mice, the tumors can be
disassociated, and the specific differentiated cell types of
interest can be selected by the usage of lineage specific markers
through the use of fluorescent activated cell sorting (FACS) or
other sorting method or by direct microdissection of tissues of
interest. These differentiated cells could then be transplanted
back to the adult animal to treat specific diseases, such as
hematopoietic disorders, endocrine deficiencies, degenerative
neurological disorders or hair loss.
[0036] (b) Selection of Model Species
[0037] Macaques and marmosets were used as exemplary species for
isolation of a primate ES cell line. Macaques, such as the rhesus
monkey, are Old World species that are the major primates used in
biomedical research. They are relatively large (about 7-10 kg).
Males take 4-5 years to mature, and females have single young.
Because of the extremely close anatomical and physiological
similarities between humans and rhesus monkeys, rhesus monkey true
ES cell lines provide a very accurate in vitro model for human
differentiation. Rhesus monkey ES cell lines and rhesus monkeys
will be particularly useful in the testing of the safety and
efficacy of the transplantation of differentiated cell types into
whole animals for the treatment of specific diseases or conditions.
In addition, the techniques developed for the rhesus ES cell lines
model the generation, characterization and manipulation of human ES
cell lines.
[0038] The common marmoset (Callithrix jacchus) is a New World
primate species with reproductive characteristics that make it an
excellent choice for ES cell derivation. Marmosets are small (about
350-400 g), have a short gestation period (144 days), reach sexual
maturity in about 18 months, and routinely have twins or triplets.
Unlike in macaques, it is possible to routinely synchronize ovarian
cycles in the marmoset with prostaglandin analogs, making
collection of age-matched embryos from multiple females possible,
and allowing efficient embryo transfer to synchronized recipients
with 70%-80% of embryos transferred resulting in pregnancies.
Because of these reproductive characteristics that allow for the
routine efficient transfer of multiple embryos, marmosets provide
an excellent primate species in which to generate transgenic models
for human diseases.
[0039] There are approximately 200 primate species in the world.
The most fundamental division that divides higher primates is
between Old World and New world species. The evolutionary distance
between the rhesus monkey and the common marmoset is far greater
than the evolutionary distance between humans and rhesus monkeys.
Because it is here demonstrated that it is possible to isolate ES
cell lines from a representative species of both the Old World and
New World group using similar conditions, the techniques described
below may be used successfully in deriving ES cell lines in other
higher primates as well. Given the close evolutionary distance
between rhesus macaques and humans, and the fact that
feeder-dependent human EC cell lines can be grown in conditions
similar to those that support primate ES cell lines, the same
growth conditions will allow the isolation and growth of human ES
cells. In addition, human ES cell lines will be permanent cell
lines that will also be distinguished from all other permanent
human cell lines by their normal karyotype and the expression of
the same combination of cell surface markers (alkaline phosphotase,
preferably SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81) that characterize
other primate ES cell lines. A normal karyotype and the expression
of this combination of cell surface markers will be defining
properties of true human ES cell lines, regardless of the method
used for their isolation and regardless of their tissue of
origin.
[0040] No other primate (human or non-human) ES cell line is known
to exist. The only published permanent, euploid, embryo-derived
cell lines that have been convincingly demonstrated to
differentiate into derivatives of all three germ layers have been
derived from rodents (the mouse, rat, and hamster), and possibly
from rabbit. The published reports of embryo-derived cell lines
from domestic species have failed to convincingly demonstrate
differentiation of derivatives of all three embryonic germ layers
or have not been permanent cell lines. Research groups in Britain
and Singapore are informally reported, later than the work
described here, to have attempted to derive human ES cell lines
from surplus in vitro fertilization-produced human embryos,
although they have not yet reported success in demonstrating
pluripotency of their cells and have failed to isolate permanent
cell lines. In the only published report on attempts to isolate
human ES cells, conditions were used (LIF in the absence of
fibroblast feeder layers) that the results below will indicate will
not result in primate ES cells which can remain in an
undifferentiated state. It is not surprising, then that the cells
grown out of human ICMs failed to continue to proliferate after 1
or 2 subcultures, Bongso et al. Hum. Reprod. 9:2100-2117
(1994).
[0041] (2) Embryonic Stem Cell Isolation
[0042] A preferable medium for isolation of embryonic stem cells is
"ES medium." ES medium consists of 80% Dulbecco's modified Eagle's
medium (DMEM; no pyruvate, high glucose formulation, Gibco BRL),
with 20% fetal bovine serum (FBS; Hyclone), 0.1 mM
.beta.-mercaptoethanol (Sigma), 1% non-essential amino acid stock
(Gibco BRL). Preferably, fetal bovine serum batches are compared by
testing clonal plating efficiency of a low passage mouse ES cell
line (ES.sub.jt3), a cell line developed just for the purpose of
this test. FBS batches must be compared because it has been found
that batches vary dramatically in their ability to support
embryonic cell growth, but any other method of assaying the
competence of FBS batches for support of embryonic cells will work
as an alternative.
[0043] Primate ES cells are isolated on a confluent layer of murine
embryonic fibroblast in the presence of ES cell medium. Embryonic
fibroblasts are preferably obtained from 12 day old fetuses from
outbred CF1 mice (SASCO), but other strains may be used as an
alternative. Tissue culture dishes are preferably treated with 0.1%
gelatin (type I; Sigma).
[0044] For rhesus monkey embryos, adult female rhesus monkeys
(greater than four years old) demonstrating normal ovarian cycles
are observed daily for evidence of menstrual bleeding (day 1 of
cycle=the day of onset of menses). Blood samples are drawn daily
during the follicular phase starting from day 8 of the menstrual
cycle, and serum concentrations of luteinizing hormone are
determined by radioimmunoassay. The female is paired with a male
rhesus monkey of proven fertility from day 9 of the menstrual cycle
until 48 hours after the luteinizing hormone surge; ovulation is
taken as the day following the luteinizing hormone surge. Expanded
blastocysts are collected by non-surgical uterine flushing at six
days after ovulation. This procedure routinely results in the
recovery of an average 0.4 to 0.6 viable embryos per rhesus monkey
per month, Seshagiri et al. Am J Primatol 29:81-91, 1993.
[0045] For marmoset embryos, adult female marmosets (greater than
two years of age) demonstrating regular ovarian cycles are
maintained in family groups, with a fertile male and up to five
progeny. Ovarian cycles are controlled by intramuscular injection
of 0.75 g of the prostaglandin PGF2a analog cloprostenol
(Estrumate, Mobay Corp, Shawnee, Kans.) during the middle to late
luteal phase. Blood samples are drawn on day 0 (immediately before
cloprostenol injection), and on days 3, 7, 9, 11, and 13. Plasma
progesterone concentrations are determined by ELISA. The day of
ovulation is taken as the day preceding a plasma progesterone
concentration of 10 ng/ml or more. At eight days after ovulation,
expanded blastocysts are recovered by a non-surgical uterine flush
procedure, Thomson et al. "Non-surgical uterine stage
preimplantation embryo collection from the common marmoset," J Med
Primatol, 23:333-336 (1994). This procedure results in the average
production of 1.0 viable embryos per marmoset per month.
[0046] The zona pellucida is removed from blastocysts by brief
exposure to pronase (Sigma). For immunosurgery, blastocysts are
exposed to a 1:50 dilution of rabbit anti-marmoset spleen cell
antiserum (for marmoset blastocysts) or a 1:50 dilution of rabbit
anti-rhesus monkey (for rhesus monkey blastocysts) in DMEM for 30
minutes, then washed for 5 minutes three times in DMEM, then
exposed to a 1:5 dilution of Guinea pig complement (Gibco) for 3
minutes.
[0047] After two further washes in DMEM, lysed trophectoderm cells
are removed from the intact inner cell mass (ICM) by gentle
pipetting, and the ICM plated on mouse inactivated (3000 rads gamma
irradiation) embryonic fibroblasts.
[0048] After 7-21 days, ICM-derived masses are removed from
endoderm outgrowths with a micropipette with direct observation
under a stereo microscope, exposed to 0.05% Trypsin-EDTA (Gibco)
supplemented with 1% chicken serum for 3-5 minutes and gently
dissociated by gentle pipetting through a flame polished
micropipette.
[0049] Dissociated cells are replated on embryonic feeder layers in
fresh ES medium, and observed for colony formation. Colonies
demonstrating ES-like morphology are individually selected, and
split again as described above. The ES-like morphology is defined
as compact colonies having a high nucleus to cytoplasm ratio and
prominent nucleoli. Resulting ES cells are then routinely split by
brief trypsinization or exposure to Dulbecco's Phosphate Buffered
Saline (without calcium or magnesium and with 2 mM EDTA) every 1-2
weeks as the cultures become dense. Early passage cells are also
frozen and stored in liquid nitrogen.
[0050] Cell lines may be karyotyped with a standard G-banding
technique (such as by the Cytogenetics Laboratory of the University
of Wisconsin State Hygiene Laboratory, which provides routine
karyotyping services) and compared to published karyotypes for the
primate species.
[0051] Isolation of ES cell lines from other primate species would
follow a similar procedure, except that the rate of development to
blastocyst can vary by a few days between species, and the rate of
development of the cultured ICMs will vary between species. For
example, six days after ovulation, rhesus monkey embryos are at the
expanded blastocyst stage, whereas marmoset embryos don't reach the
same stage until 7-8 days after ovulation. The Rhesus ES cell lines
were obtained by splitting the ICM-derived cells for the first time
at 7-16 days after immunosurgery; whereas the marmoset ES cells
were derived with the initial split at 7-10 days after
immunosurgery. Because other primates also vary in their
developmental rate, the timing of embryo collection, and the timing
of the initial ICM split will vary between primate species, but the
same techniques and culture conditions will allow ES cell
isolation.
[0052] Because ethical considerations in the U.S. do not allow the
recovery of human in vivo fertilized preimplantation embryos from
the uterus, human ES cells that are derived from preimplantation
embryos will be derived from in vitro fertilized (IVF) embryos.
Experiments on unused (spare) human IVF-produced embryos are
allowed in many countries, such as Singapore and the United
Kingdom, if the embryos are less than 14 days old. Only high
quality embryos are suitable for ES isolation. Present defined
culture conditions for culturing the one cell human embryo to the
expanded blastocyst are suboptimal but practicable, Bongso et al.,
Hum Reprod 4:706-713, 1989. Co-culturing of human embryos with
human oviductal cells results in the production of high blastocyst
quality. IVF-derived expanded human blastocysts grown in cellular
co-culture, or in improved defined medium, will allow the isolation
of human ES cells with the same procedures described above for
nonhuman primates.
[0053] (3) Defining Characteristics of Primate ES Cells
[0054] Primate embryonic stem cells share features with the primate
ICM and with pluripotent human embryonal carcinoma cells. Putative
primate ES cells may therefore be characterized by morphology and
by the expression of cell surface markers characteristic of human
EC cells. Additionally, putative primate ES cells may be
characterized by developmental potential, karyotype and
immortality.
[0055] (a) Morphology
[0056] The colony morphology of primate embryonic stem cell lines
is similar to, but distinct from, mouse embryonic stem cells. Both
mouse and primate ES cells have the characteristic features of
undifferentiated stem cells, with high nuclear/cytoplasmic ratios,
prominent nucleoli, and compact colony formation. The colonies of
primate ES cells are flatter than mouse ES cell colonies and
individual primate ES cells can be easily distinguished. In FIG. 2,
reference character A indicates a phase contrast photomicrograph of
cell line R278.5 demonstrating the characteristic primate ES cell
morphology.
[0057] (b) Cell Surface Markers
[0058] A primate ES cell line of the present invention is distinct
from mouse ES cell lines by the presence or absence of the cell
surface markers described below.
[0059] One set of glycolipid cell surface markers is known as the
Stage-specific embryonic antigens 1 through 4. These antigens can
be identified using antibodies for SSEA 1, preferably SSEA-3 and
SSEA-4 which are available from the Developmental Studies Hybridoma
Bank of the National Institute of Child Health and Human
Development. The cell surface markers referred to as TRA-1-60 and
TRA-1-81 designate antibodies from hybridomas developed by Peter
Andrews of the University of Sheffield and are described in Andrews
et al., "Cell lines from human germ cell tumors," In: Robertson E,
ed. Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach. Oxford: IRL Press, 207-246, 1987. The antibodies were
localized with a biotinylated secondary antibody and then an
avidin/biotinylated horseradish peroxidase complex (Vectastain ABC
System, Vector Laboratories). Alternatively, it should also be
understood that other antibodies for these same cell surface
markers can be generated. NTERA-2 cl. D1, a pluripotent human EC
cell line (gift of Peter Andrews), may be used as a negative
control for SSEA-1, and as a positive control for SSEA-3, SSEA-4,
TRA-1-60, and TRA-1-81. This cell line was chosen for positive
control only because it has been extensively studied and reported
in the literature, but other human EC cell lines may be used as
well.
[0060] Mouse ES cells (ES.sub.jt3) are used as a positive control
for SSEA-1, and for a negative control for SSEA-3, SSEA-4,
TRA-1-60, and TRA-1-81. Other routine negative controls include
omission of the primary or secondary antibody and substitution of a
primary antibody with an unrelated specificity.
[0061] Alkaline phosphatase may be detected following fixation of
cells with 4% para-formaldehyde using "Vector Red" (Vector
Laboratories) as a substrate, as described by the manufacturer
(Vector Laboratories). The precipitate formed by this substrate is
red when viewed with a rhodamine filter system, providing
substantial amplification over light microscopy.
[0062] Table 1 diagrams a comparison of mouse ES cells, primate ES
cells, and human EC cells. The only cells reported to express the
combination of markers SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 other
than primate ES cells are human EC cells. The globo-series
glycolipids SSEA-3 and SSEA-4 are consistently present on human EC
cells, and are of diagnostic value in distinguishing human EC cell
tumors from human yolk sac carcinomas, choriocarcinomas, and other
lineages which lack these markers, Wenk et al., Int J Cancer
58:108-115, 1994. A recent survey found SSEA-3 and SSEA-4 to be
present on all of over 40 human EC cell lines examined, Wenk et al.
TRA-1-60 and TRA-1-81 antigens have been studied extensively on a
particular pluripotent human EC cell line, NTERA-2 CL. D1, Andrews
et al, supra. Differentiation of NTERA-2 CL. D1 cells in vitro
results in the loss of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81
expression and the increased expression of the lacto-series
glycolipid SSEA-1, Andrews et al, supra. This contrasts with
undifferentiated mouse ES cells, which express SSEA-1, and neither
SSEA-3 nor SSEA-4. Although the function of these antigens are
unknown, their shared expression by R278.5 cells and human EC cells
suggests a close embryological similarity. Alkaline phosphatase
will also be present on all primate ES cells. A successful primate
ES cell culture of the present invention will correlate with the
cell surface markers found in the rhesus macaque and marmoset cell
lines described in Table 1.
[0063] As disclosed below in Table 1, the rhesus macaque and
marmoset cell lines are identical to human EC cell lines for the 5
described markers. Therefore, a successful primate ES cell culture
will also mimic human EC cells. However, there are other ways to
discriminate ES cells from EC cells. For example, the primate ES
cell line has a normal karyotype and the human EC cell line is
aneuploid.
[0064] In FIG. 3, the photographs labelled A through F demonstrate
the characteristic staining of these markers on a rhesus monkey ES
cell line designated R278.5.
1 TABLE 1 Mouse C. jacchus M. mulatta Human EC ES ES ES (NTERA-2
cl.D1) SSEA-1 + - - - SSEA-3 - + + + SSEA-4 - + + + Tra-1-60 - + +
+ Tra-1-81 - + + +
[0065] (c) Developmental Potential
[0066] Primate ES cells of the present invention are pluripotent.
By "pluripotent" we mean that the cell has the ability to develop
into any cell derived from the three main germ cell layers or an
embryo itself. When injected into SCID mice, a successful primate
ES cell line will differentiate into cells derived from all three
embryonic germ layers including: bone, cartilage, smooth muscle,
striated muscle, and hematopoietic cells (mesoderm); liver,
primitive gut and respiratory epithelium (endoderm); neurons, glial
cells, hair follicles, and tooth buds (ectoderm).
[0067] This experiment can be accomplished by injecting
approximately 0.5-1.0.times.10.sup.6 primate ES cells into the rear
leg muscles of 8-12 week old male SCID mice. The resulting tumors
can be fixed in 4% paraformaldehyde and examined histologically
after paraffin embedding at 8-16 weeks of development. In FIG. 4,
photomicrographs designated A-F are of sections of tumors formed by
injection of rhesus ES cells into the hind leg muscles of SCID mice
and analyzed 15 weeks later demonstrating cartilage, smooth muscle,
and striated muscle (mesoderm); stratified squamous epithelium with
hair follicles, neural tube with ventricular, intermediate, and
mantle layers (ectoderm); ciliated columnar epithelium and villi
lined by absorptive enterocytes and mucus-secreting goblet cells
(endoderm).
[0068] A successful nonhuman primate ES cell line will have the
ability to participate in normal development when combined in
chimeras with normal preimplantation embryos. Chimeras between
preimplantation nonhuman primate embryos and nonhuman primate ES
cells can be formed by routine methods in several ways. (i)
injection chimeras: 10-15 nonhuman primate ES cells can be
microinjected into the cavity of an expanded nonhuman primate
blastocyst; (ii) aggregation chimeras: nonhuman primate morulae can
be co-cultured on a lawn of nonhuman primate ES cells and allowed
to aggregate; and (iii) tetraploid chimeras: 10-15 nonhuman primate
ES cells can be aggregated with tetraploid nonhuman primate morulae
obtained by electrofusion of 2-cell embryos, or incubation of
morulae in the cytoskeletal inhibitor cholchicine. The chimeras can
be returned to the uterus of a female nonhuman primate and allowed
to develop to term, and the ES cells will contribute to normal
differentiated tissues derived from all three embryonic germ layers
and to germ cells. Because nonhuman primate ES can be genetically
manipulated prior to chimera formation by standard techniques,
chimera formation followed by embryo transfer can lead to the
production of transgenic nonhuman primates.
[0069] (d) Karyotype
[0070] Successful primate ES cell lines have normal karyotypes.
Both XX and XY cells lines will be derived. The normal karyotypes
in primate ES cell lines will be in contrast to the abnormal
karyotype found in human embryonal carcinoma (EC), which are
derived from spontaneously arising human germ cell tumors
(teratocarcinomas). Human embryonal carcinoma cells have a limited
ability to differentiate into multiple cell types and represent the
closest existing cell lines to primate ES cells. Although
tumor-derived human embryonal carcinoma cell lines have some
properties in common with embryonic stem cell lines, all human
embryonal carcinoma cell lines derived to date are aneuploid. Thus,
primate ES cell lines and human EC cell lines can be distinguished
by the normal karyotypes found in primate ES cell lines and the
abnormal karyotypes found in human EC lines. By "normal karyotype"
it is meant that all chromosomes normally characteristic of the
species are present and have not been noticeably altered.
[0071] Because of the abnormal karyotypes of human embryonal
carcinoma cells, it is not clear how accurately their
differentiation reflects normal differentiation. The range of
embryonic and extraembryonic differentiation observed with primate
ES cells will typically exceed that observed in any human embryonal
carcinoma cell line, and the normal karyotypes of the primate ES
cells suggests that this differentiation accurately recapitulates
normal differentiation.
[0072] (e) Immortality
[0073] Immortal cells are capable of continuous indefinite
replication in vitro. Continued proliferation for longer than one
year of culture is a sufficient evidence for immortality, as
primary cell cultures without this property fail to continuously
divide for this length of time (Freshney, Culture of animal cells.
New York: Wiley-Liss, 1994). Primate ES cells will continue to
proliferate in vitro with the culture conditions described above
for longer than one year, and will maintain the developmental
potential to contribute all three embryonic germ layers. This
developmental potential can be demonstrated by the injection of ES
cells that have been cultured for a prolonged period (over a year)
into SCID mice and then histologically examining the resulting
tumors. Although karyotypic changes can occur randomly with
prolonged culture, some primate ES cells will maintain a normal
karyotype for longer than a year of continuous culture.
[0074] (f) Culture Conditions
[0075] Growth factor requirements to prevent differentiation are
different for the primate ES cell line of the present invention
than the requirements for mouse ES cell lines. In the absence of
fibroblast feeder layers, Leukemia inhibitory factor (LIF) is
necessary and sufficient to prevent differentiation of mouse ES
cells and to allow their continuous passage. Large concentrations
of cloned LIF fail to prevent differentiation of primate ES cell
lines in the absence of fibroblast feeder layers. In this regard,
primate ES stem cells are again more similar to human EC cells than
to mouse ES cells, as the growth of feeder-dependent human EC cells
lines is not supported by LIF in the absence of fibroblasts.
[0076] (g) Differentiation to Extra Embryonic Tissues
[0077] When grown on embryonic fibroblasts and allowed to grow for
two weeks after achieving confluence (i.e., continuously covering
the culture surface), primate ES cells of the present invention
spontaneously differentiate and will produce chorionic
gonadotropin, indicating trophoblast differentiation (a component
of the placenta) and produce .alpha.-fetoprotein, indicating
endoderm differentiation. Chorionic gonadotropin activity can be
assayed in the medium conditioned by differentiated cells by Leydig
cell bioassay, Seshagiri & Hearn, Hum Reprod 8:279-287, 1992.
For mRNA analysis, RNA can be prepared by guanidine
isothiocyanate-phenol/chloroform extraction (1) from approximately
0.2.times.10.sup.6 differentiated cells and from 0.2.times.10.sup.6
undifferentiated cells. The relative levels of the mRNA for
.alpha.-fetoprotein and the .alpha.- and .beta.-subunit of
chorionic gonadotropin relative to glyceraldehyde-3-phosphate
dehydrogenase can be determined by semi-quantitative Reverse
Transcriptase-Polymerase Chain Reaction (RT-PCR). The PCR primers
for glyceraldehyde 3-phosphate dehydrogenase (G3PDH), obtained from
Clontech (Palo Alto, Calif.), are based on the human cDNA sequence,
and do not amplify mouse G3PDH mRNA under our conditions. Primers
for the .alpha.-fetoprotein mRNA are based on the human sequence
and flank the 7th intron (5' primer=(5') GCTGGATTGTCTGCAGGATGGGGAA
(SEQ ID NO: 1); 3' primer=(5') TCCCCTGAAGAAAATTGGTTAAAAT (SEQ ID
NO: 2)). They amplify a cDNA of 216 nucleotides. Primers for the
.beta.-subunit of chorionic gonadotropin flank the second intron
(5' primer=(5') ggatc CACCGTCAACACCACCATCTGTGC (SEQ ID NO: 3); 3'
primer=(5') ggatc CACAGGTCAAAGGGTGGTCCTTGGG (SEQ ID NO: 4))
(nucleotides added to the hCGb sequence to facilitate sub-cloning
are shown in lower case italics). They amplify a cDNA of 262 base
pairs. The primers for the CG.alpha. subunit can be based on
sequences of the first and fourth exon of the rhesus gene (5'
primer=(5') gggaattc GCAGTTACTGAGAACTCACAAG (SEQ ID NO: 5); 3'
primer=(5') gggaattc GAAGCATGTCAAAGTGGTATGG (SEQ ID NO: 6)) and
amplify a cDNA of 556 base pairs. The identity of the
.alpha.-fetoprotein, CG.alpha. and CG.beta. cDNAs can be verified
by subcloning and sequencing.
[0078] For Reverse Transcriptase-Polymerase Chain Reaction
(RT-PCR), 1 to 5 .mu.l of total R278.5 RNA can be reverse
transcribed as described Golos et al. Endocrinology
133(4):1744-1752, 1993, and one to 20 .mu.l of reverse
transcription reaction was then subjected to the polymerase chain
reaction in a mixture containing 1-12.5 pmol of each G3PDH primer,
10-25 pmol of each mRNA specific primer, 0.25 mM dNTPs (Pharmacia,
Piscataway, N.J.), 1X AmpliTaq buffer (final reaction
concentrations=10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001%
(w/v) gelatin) 2.5 .mu.Ci of deoxycytidine 5'a[32P]triphosphate
(DuPont, Boston, Mass.), 10% glycerol and 1.25 U of AmpliTaq
(Perkin-Elmer, Oak Brook, Ill.) in a total volume of 50 .mu.l. The
number of amplification rounds which produced linear increases in
target cDNAs and the relation between input RNA and amount of PCR
product is empirically determined as by Golos et al. Samples were
fractionated in 3% Nusieve (FMC, Rockland, Me.) agarose gels (1X
TBE running buffer) and DNA bands of interest were cut out, melted
at 65.degree. C. in 0.5 ml TE, and radioactivity determined by
liquid scintillation counting. The ratio of counts per minute in a
specific PCR product relative to cpm of G3PDH PCR product is used
to estimate the relative levels of a mRNAs among differentiated and
undifferentiated cells.
[0079] The ability to differentiate into trophectoderm in vitro and
the ability of these differentiated cells to produce chorionic
gonadotropin distinguishes the primate ES cell line of the present
invention from all other published ES cell lines.
EXAMPLES
[0080] (1) Animals and Embryos
[0081] As described above, we have developed a technique for
non-surgical, uterine-stage embryo recovery from the rhesus macaque
and the common marmoset.
[0082] To supply rhesus embryos to interested investigators, The
Wisconsin Regional Primate Research Center (WRPRC) provides a
preimplantation embryo recovery service for the rhesus monkey,
using the non-surgical flush procedure described above. During
1994, 151 uterine flushes were attempted from rhesus monkeys,
yielding 80 viable embryos (0.53 embryos per flush attempt).
[0083] By synchronizing the reproductive cycles of several
marmosets, significant numbers of in vivo produced, age-matched,
preimplantation primate embryos were studied in controlled
experiments for the first time. Using marmosets from the
self-sustaining colony (250 animals) of the Wisconsin Regional
Primate Research Center (WRPRC), we recovered 54 viable morulae or
blastocysts, 7 unfertilized oocytes or degenerate embryos, and 5
empty zonae pellucidae in a total of 54 flush attempts (1.0 viable
embryo-flush attempt). Marmosets have a 28 day ovarian cycle, and
because this is a non-surgical procedure, females can be flushed on
consecutive months, dramatically increasing the embryo yield
compared to surgical techniques which require months of rest
between collections.
[0084] (2) Rhesus Macaque Embryonic Stem Cells
[0085] Using the techniques described above, we have derived three
independent embryonic stem cell lines from two rhesus monkey
blastocysts (R278.5, R366, and R367). One of these, R278.5, remains
undifferentiated and continues to proliferate after continuous
culture for over one year. R278.5 cells have also been frozen and
successfully thawed with the recovery of viable cells.
[0086] The morphology and cell surface markers of R278.5 cells are
indistinguishable from human EC cells, and differ significantly
from mouse ES cells. R278.5 cells have a high nucleus/cytoplasm
ratio and prominent nucleoli, but rather than forming compact,
piled-up colonies with indistinct cell borders similar to mouse ES
cells, R278.5 cells form flatter colonies with individual, distinct
cells (FIG. 2 A). R278.5 cells express the SSEA-3, SSEA-4,
TRA-1-60, and TRA-81 antigens (FIG. 3 and Table 1), none of which
are expressed by mouse ES cells. The only cells known to express
the combination of markers SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81
other than primate ES cells are human EC cells. The globo-series
glycolipids SSEA-3 and SSEA-4 are consistently present on human EC
cells, and are of diagnostic value in distinguishing human EC cell
tumors from yolk sac carcinomas, choriocarcinomas and other stem
cells derived from human germ cell tumors which lack these markers,
Wenk et al, Int J Cancer 58:108-115, 1994. A recent survey found
SSEA-3 and SSEA-4 to be present on all of over 40 human EC cell
lines examined (Wenk et al.).
[0087] TRA-1-60 and TRA-1-81 antigens have been studied extensively
on a particular pluripotent human EC cell line, NTERA-2 CL. D1
(Andrews et al.). Differentiation of NTERA-2 CL. D1 cells in vitro
results in the loss of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81
expression and the increased expression of the lacto-series
glycolipid SSEA-1. Undifferentiated mouse ES cells, on the other
hand, express SSEA-1, and not SSEA-3, SSEA-4, TRA-1-60 or TRA-1-81
(Wenk et al.). Although the function of these antigens is unknown,
their expression by R278.5 cells suggests a close embryological
similarity between primate ES cells and human EC cells, and
fundamental differences between primate ES cells and mouse ES
cells.
[0088] R278.5 cells also express alkaline phosphatase. The
expression of alkaline phosphatase is shared by both primate and
mouse ES cells, and relatively few embryonic cells express this
enzyme. Positive cells include the ICM and primitive ectoderm
(which are the most similar embryonic cells in the intact embryo to
ES cells), germ cells, (which are totipotent), and a very limited
number of neural precursors, Kaufman M H. The atlas of mouse
development. London: Academic Press, 1992. Cells not expressing
this enzyme will not be primate ES cells.
[0089] Although cloned human LIF was present in the medium at cell
line derivation and for initial passages, R278.5 cells grown on
mouse embryonic fibroblasts without exogenous LIF remain
undifferentiated and continued to proliferate. R278.5 cells plated
on gelatin-treated tissue culture plates without fibroblasts
differentiated to multiple cell types or failed to attach and died,
regardless of the presence or absence of exogenously added human
LIF (FIG. 2). Up to 10.sup.4 units/ml human LIF fails to prevent
differentiation. In addition, added LIF fails to increase the
cloning efficiency or proliferation rate of R278.5 cells on
fibroblasts. Since the derivation of the R278.5 cell line, we have
derived two additional rhesus ES cell lines (R366 and R367) on
embryonic fibroblasts without any exogenously added LIF at initial
derivation. R366 and R367 cells, like R278.5 cells, continue to
proliferate on embryonic fibroblasts without exogenously added LIF
and differentiate in the absence of fibroblasts, regardless of the
presence of added LIF. RT-PCR performed on mRNA from spontaneously
differentiated R278.5 cells revealed .alpha.-fetoprotein mRNA (FIG.
4). .alpha.-fetoprotein is a specific marker for endoderm, and is
expressed by both extraembryonic (yolk sac) and embryonic (fetal
liver and intestines) endoderm-derived tissues. Epithelial cells
resembling extraembryonic endoderm are present in cells
differentiated in vitro from R278.5 cells (FIG. 2). Bioactive CG
(3.89 mI units/ml) was present in culture medium collected from
differentiated cells, but not in medium collected from
undifferentiated cells (less than 0.03 mI units/ml), indicating the
differentiation of trophoblast, a trophectoderm derivative. The
relative level of the CG.alpha. mRNA increased 23.9-fold after
differentiation (FIG. 4).
[0090] All SCID mice injected with R278.5 cells in either
intra-muscular or intra-testicular sites formed tumors, and tumors
in both sites demonstrated a similar range of differentiation. The
oldest tumors examined (15 weeks) had the most advanced
differentiation, and all had abundant, unambiguous derivatives of
all three embryonic germ layers, including gut and respiratory
epithelium (endoderm); bone, cartilage, smooth muscle, striated
muscle (mesoderm); ganglia, glia, neural precursors, and stratified
squamous epithelium (ectoderm), and other unidentified cell types
(FIG. 5). In addition to individual cell types, there was organized
development of some structures which require complex interactions
between different cell types. Such structures included gut lined by
villi with both absorptive enterocytes and mucus-secreting goblet
cells, and sometimes encircled by layers of smooth muscle in the
same orientation as muscularis mucosae (circular) and muscularis
(outer longitudinal layer and inner circular layer); neural tubes
with ventricular, intermediate, and mantle layers; and hair
follicles with hair shafts (FIG. 5).
[0091] The essential characteristics that define R278.5 cells as ES
cells include: indefinite (greater than one year) undifferentiated
proliferation in vitro, normal karyotype, and potential to
differentiate to derivatives of trophectoderm and all three
embryonic germ layers. In the mouse embryo, the last cells capable
of contributing to derivatives of both trophectoderm and ICM are
early ICM cells. The timing of commitment to ICM or trophectoderm
has not been established for any primate species, but the potential
of rhesus ES cells to contribute to derivatives of both suggests
that they most closely resemble early totipotent embryonic cells.
The ability of rhesus ES cells to form trophoblast in vitro
distinguishes primate ES cell lines from mouse ES cells. Mouse ES
cell have not been demonstrated to form trophoblast in vitro, and
mouse trophoblast does not produce gonadotropin. Rhesus ES cells
and mouse ES cells do demonstrate the similar wide range of
differentiation in tumors that distinguishes ES cells from EC
cells. The development of structures composed of multiple cell
types such as hair follicles, which require inductive interactions
between the embryonic epidermis and underlying mesenchyme,
demonstrates the ability of rhesus ES cells to participate in
complex developmental processes.
[0092] The rhesus ES lines R366 and R367 have also been further
cultured and analyzed. Both lines have a normal XY karyotype and
were proliferated in an undifferentiated state for about three
months prior to freezing for later analysis. Samples of each of the
cell lines R366 and R367 were injected into SCID mice which then
formed teratomas identical to those formed by R278.5 cells. An
additional rhesus cell line R394 having a normal XX karyotype was
also recovered. All three of these cell lines, R366, R367 and R394
are identical in morphology, growth characteristics, culture
requirements and in vitro differentiation characteristics, i.e. the
trait of differentiation to multiple cell types in the absence of
fibroblasts, to cell line 278.5.
[0093] It has been determined that LIF is not required either to
derive or proliferate these ES cultures. Each of the cell lines
R366, R367 and R394 were derived and cultured without exogenous
LIF.
[0094] It has also been demonstrated that the particular source of
fibroblasts for co-culture is not critical. Several fibroblast cell
lines have been tested both with rhesus line R278.5 and with the
marmoset cell lines described below. The fibroblasts tested include
mouse STO cells (ATCC 56-X), mouse 3T3 cells (ATCC 48-X), primary
rhesus monkey embryonic fibroblasts derived from 36 day rhesus
fetuses, and mouse Sl/Sl.sup.4 cells, which are deficient in the
steel factor. All these fibroblast cell lines were capable of
maintaining the stem cell lines in an undifferentiated state. Most
rapid proliferation of the stem cells was observed using primary
mouse embryonic fibroblasts.
[0095] Unlike mouse ES cells, neither rhesus ES cells nor
feeder-dependent human EC cells remain undifferentiated and
proliferate in the presence of soluble human LIF without
fibroblasts. The factors that fibroblasts produce that prevent the
differentiation of rhesus ES cells or feeder-dependent human EC
cells are unknown, but the lack of a dependence on LIF is another
characteristic that distinguishes primate ES cells from mouse ES
cells. The growth of rhesus monkey ES cells in culture conditions
similar to those required by feeder-dependent human EC cells, and
the identical morphology and cell surface markers of rhesus ES
cells and human EC cells, suggests that similar culture conditions
will support human ES cells.
[0096] Rhesus ES cells will be important for elucidating the
mechanisms that control the differentiation of specific primate
cell types. Given the close evolutionary distance and the
developmental and physiological similarities between humans and
rhesus monkeys, the mechanisms controlling the differentiation of
rhesus cells will be very similar to the mechanisms controlling the
differentiation of human cells. The importance of elucidating these
mechanisms is that once they are understood, it will be possible to
direct primate ES cells to differentiate to specific cell types in
vitro, and these specific cell types can be used for
transplantation to treat specific diseases.
[0097] Because ES cells have the developmental potential to give
rise to any differentiated cell type, any disease that results in
part or in whole from the failure (either genetic or acquired) of
specific cell types will be potentially treatable through the
transplantation of cells derived from ES cells. Rhesus ES cells and
rhesus monkeys will be invaluable for testing the efficacy and
safety of the transplantation of specific cell types derived from
ES cells. A few examples of human diseases potentially treatable by
this approach with human ES cells include degenerative neurological
disorders such as Parkinson's disease (dopanergic neurons),
juvenile onset diabetes (pancreatic .beta.-islet cells) or Acquired
Immunodeficiency Disease (lymphocytes). Because undifferentiated ES
cells can proliferate indefinitely in vitro, they can be
genetically manipulated with standard techniques either to prevent
immune rejection after transplantation, or to give them new genetic
properties to combat specific diseases. For specific cell types
where immune rejection can be prevented, cells derived from rhesus
monkey ES cells or other non-human primate ES cells could be used
for transplantation to humans to treat specific diseases.
[0098] (3) Marmoset Embryonic Stem Cells
[0099] Our method for creating an embryonic stem cell line is
described above. Using isolated ICM's derived by immunosurgery from
marmoset blastocysts, we have isolated 7 putative ES cell lines,
each of which have been cultured for over 6 months.
[0100] One of these, Cj11, was cultured continuously for over 14
months, and then frozen for later analysis. The Cj11 cell line and
other marmoset ES cell lines have been successfully frozen and then
thawed with the recovery of viable cells. These cells have a high
nuclear/cytoplasmic ratio, prominent nucleoli, and a compact colony
morphology similar to the pluripotent human embryonal carcinoma
(EC) cell line NT2/D2.
[0101] Four of the cell lines we have isolated have normal XX
karyotypes, and one has a normal XY karyotype (Karyotypes were
performed by Dr. Charles Harris, University of Wisconsin). These
cells were positive for a series of cell surface markers (alkaline
phosphatase, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) that in
combination are definitive markers for undifferentiated human
embryonal carcinoma cells (EC) cells and primate ES cells. In
particular, these markers distinguish EC cells from the earliest
lineages to differentiate in the human preimplantation embryo,
trophectoderm (represented by BeWO choriocarcinoma cells) and
extraembryonic endoderm (represented by 1411H yolk sac carcinoma
cells).
[0102] When the putative marmoset ES cells were removed from
fibroblast feeders, they differentiated into cells of several
distinct morphologies. Among the differentiated cells,
trophectoderm is indicated by the secretion of chorionic
gonadotropin and the presence of the chorionic gonadotropin
.beta.-subunit mRNA. 12.7 mIU/ml luteinizing hormone (LH) activity
was measured in the WRPRC core assay lab using a mouse Leydig cell
bioassay in medium conditioned 24 hours by putative ES cells
allowed to differentiate for one week. Note that chorionic
gonadotrophin has both LH and FSH activity, and is routinely
measured by LH assays. Control medium from undifferentiated ES
cells had less than 1 mIU/ml LH activity.
[0103] Chorionic gonadotropin .beta.-subunit mRNA was detected by
reverse transcriptase-polymerase chain reaction (RT-PCR). DNA
sequencing confirmed the identity of the chorionic gonadotrophin
.beta.-subunit.
[0104] Endoderm differentiation (probably extraembryonic endoderm)
was indicated by the presence of .alpha.-fetoprotein mRNA, detected
by RT-PCR.
[0105] When the marmoset ES cells were grown in high densities,
over a period of weeks epithelial cells differentiated and covered
the culture dish. The remaining groups of undifferentiated cells
rounded up into compact balls and then formed embryoid bodies (as
shown in FIG. 6) that recapitulated early development with
remarkable fidelity. Over 3-4 weeks, some of the embryoid bodies
formed a bilaterally symmetric pyriform embryonic disc, an amnion,
a yolk sac, and a mesoblast outgrowth attaching the caudal pole of
the amnion to the culture dish.
[0106] Histological and ultrastructural examination of one of these
embryoid bodies (formed from a cell line that had been passaged
continuously for 6 months) revealed a remarkable resemblance to a
stage 6-7 post-implantation embryo. The embryonic disc was composed
of a polarized, columnar epithelial epiblast (primitive ectoderm)
layer separated from a visceral endoderm (primitive endoderm)
layer. Electron microscopy of the epiblast revealed apical
junctional complexes, apical microvilli, subapical intermediate
filaments, and a basement membrane separating the epiblast from
underlying visceral endoderm. All of these elements are features of
the normal embryonic disc. In the caudal third of the embryonic
disc, there was a midline groove, disruption of the basement
membrane, and mixing of epiblast cells with underlying endodermal
cells (early primitive streak). The amnion was composed of an inner
squamous (ectoderm) layer continuous with the epiblast and an outer
mesoderm layer. The bilayered yolk sac had occasional
endothelial-lined spaces containing possible hematopoietic
precursors.
[0107] The morphology, immortality, karyotype, and cell surface
markers of these marmoset cells identify these marmoset cells as
primate ES cells similar to the rhesus ES cells. Since the last
cells in the mammalian embryo capable of contributing to both
trophectoderm derivatives and endoderm derivatives are the
totipotent cells of the early ICM, the ability of marmoset ES cells
to contribute to both trophoblast and endoderm demonstrates their
similarities to early totipotent embryonic cells of the intact
embryo. The formation of embryoid bodies by marmoset ES cells, with
remarkable structural similarities to the early post-implantation
primate embryo, demonstrates the potential of marmoset ES cells to
participate in complex developmental processes requiring the
interaction of multiple cell types.
[0108] Given the reproductive characteristics of the common
marmoset described above (efficient embryo transfer, multiple
young, short generation time), marmoset ES cells will be
particularly useful for the generation of transgenic primates.
Although mice have provided invaluable insights into gene function
and regulation, the anatomical and physiological differences
between humans and mice limit the usefulness of transgenic mouse
models of human diseases. Transgenic primates, in addition to
providing insights into the pathogenesis of specific diseases, will
provide accurate animal models to test the efficacy and safety of
specific treatments.
Sequence CWU 1
1
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