Title of Invention

METHOD OF MAKING EMBRYOID BODIES FROM PRIMATE EMBRYONIC STEM CELLS.

Abstract PRIMATE EMBRYOID BODIES ARE FORMED FROM PRIMATE ES CELLS. THE ES CELLS FROM CLUMPS. ONE THEN REMOVES THE CLUMPS, AS CLUMPS, AND PERMITS INCUBATION UNDER NON-ADHERENT CONDITIONS. THE DEVELOPMENT OF EMBRYOID BODIES FROM PRIMATE ES CELLS IS DEPENDENT ON MAINTAINING THE AGGREGATION OF CELLS, AS INDIVIDUALIZED CELLS WILL RAPIDLY DIE.
Full Text METHOD OF MAKING EMBRYOID BODIES FROM
PRIMATE EMBRYONIC STEM CELLS
CROSS REFERENCES TO RELATED APPLICATIONS
Not applicable.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
The work with the non-human primate cells described
below was supported by a grant with United States
government support awarded by the following agency: NIH
RR11571. The United States has certain rights in this
invention. No U.S. government funds were used for the
work with human cells described herein.
BACKGROUND OF THE INVENTION
Undifferentiated primate embryonic stem ("ES") cells
can be cultured indefinitely and yet maintain the
potential to form differentiated cells of the body. See
U.S. patent 5,843,780; J. Thomson, et al., 282 Science
1145-1147 (1998); and J. Thomson, et al., 38 Biology 133-
165 (1998). The disclosure of these publications and of
all other publications referred to herein are
incorporated by reference as if fully set forth herein.
Primate ES cells thus provide an exciting new model
for understanding the differentiation and function of
human tissue, and offer new strategies for drug discovery
and testing. They also promise new therapies based on
the transplantation of ES cell-derived tissues. For
example, human and rhesus monkey ES cells injected into
immunocompromised mice form benign teratomas with
advanced differentiated derivatives representing all
three embryonic germ layers. Easily identified
differentiated cells in human ES cell teratomas include
smooth muscle, striated muscle, bone, cartilage, gut and
respiratory epithelium, keratinizing squamous epithelium,
hair, neural epithelium, and ganglia.
Human and non-human primate ES cell lines provide a
particularly powerful new model for understanding normal
human development and thus also for understanding

abnormal human development. Because of the potential
risk to the resulting child, experimental manipulation of
the post-implantation human embryo is ethically
unacceptable and as a result functional studies on human
embryos are lacking. Consequently, what is known about
human development in the early post-implantation period
is based almost entirely on static histological sections
of a few human embryos and on analogy to experimental
embryology studies of the mouse.
However, early mouse and primate development differ
significantly. For example, human and mouse embryos
differ in the timing of embryonic genome expression, in
the formation, structure, and function of the fetal
membranes and placenta and in the formation of an
embryonic disc instead of an egg cylinder. The earliest
events of human development are critically involved in
human infertility, pregnancy loss, and birth defects.
Primate ES cells offer a new window for understanding
these early human developmental events and for
understanding the pathogenesis of developmental failures.
Primate ES cells also provide a potentially
unlimited source of differentiated, euploid, non-
transformed cells for investigators interested in the
normal function and pathology of specific differentiated
primate cells. Such purified populations of specific ES
cell-derived cells will also likely be useful for drug
discovery, toxicity screens, and will provide a source of
cells for transplantation.
For tissues such as the heart that completely lack a
tissue-specific stem cell, primate ES cells will prove
even more valuable. Primate ES cells also offer the
promise of new transplantation therapies. When disease
results from the destruction or dysfunction of a limited
number of cell types, such as in Parkinson"s disease
(dopaminergic neurons), or juvenile onset diabetes

mellitus (pancreatic ?-islet cells) , the replacement of
those specific cell types by ES cell derivatives could
offer potentially life long treatment.
To accomplish these goals, it is desirable to more
efficiently differentiate ES cells to specific lineages.
Considerable progress in causing non-primate ES cell
differentiation to neural, hematopoietic, and cardiac
tissue has been made. See e.g. T. Doetschman, et al. , 87
J. Embry. And Exper. Morph. 27-45 (1985); G. Keller, 7
Current Op. In Cell Biol. 862-869 (1995); U.S. patent
5,914,2 68. In each of these examples, ES cells were
first formed into "embryoid bodies", three-dimensional ES
cell aggregates that facilitate subsequent
differentiation.
However, analogous experiments on primate ES cells
demonstrated that embryoid body formation by conventional
murine protocols fail. In such conventional protocols ES
cells are dispersed to single cells, and either allowed
to aggregate into embryoid bodies under conditions that
prevent cell attachment to the substrate, or the ES cells
are allowed to grow into embryoid bodies from single
cells or clusters suspended in methylcellulose. We have
learned that primate ES cells die rapidly when dispersed
to single cells if attachment is prevented, so they do
not successfully aggregate, and they therefore do not
grow out from clones in methylcellulose.
It can therefore be seen that a need exists for
improved methods for producing primate embryoid bodies,
and differentiated cells derived therefrom.
BRIEF SUMMARY OF THE INVENTION
The present invention provides a method for
producing primate embryoid bodies from colonies of
primate embryonic stem cells that are adhering to a
substrate. One removes the adhering colonies of the
embryonic stem cells from the substrate in clumps. One

then incubates the clumps in a container under conditions
in which the clumps are essentially inhibited from
attaching to the container and coalesce into embryoid
bodies. For purposes of this application, a clump is a
grouping of two or more stem cells, preferably a clump
large enough to be visible to the naked eye.
In one preferred form the removal step is in the
presence of an agent that promotes disassociation of the
clumps from the substrate as clumps. A purely chemical
agent such as Versene® calcium disodium EDTA chelating
agent can be used. However, more preferred is a
proteinase that preferentially acts on the extra cellular
matrix such as dispase, collagenase, catalase,
neuraminidase, pancreatin, pancreatic elastase or
trypsin. If trypsin is used the removal step must be
conducted quickly and at relatively low concentrations in
order to prevent the trypsin from also destroying the
clumps. Enzyme EDTA mixes can also be used to advantage.
In another form the removal step involves
mechanically scraping the clumps from the substrate as
clumps.
In another aspect the incubation step can be
conducted by agitating the container (e.g. by gently
rocking, shaking, or vibrating it), the container for the
incubation step can be a non-attaching bacterial grade
culture plastic, and/or the incubation step can be in the
presence of a serum-free medium which lacks serum
attachment factors.
In another aspect the invention provides primate
embryoid bodies that have, been derived (directly or
indirectly) using the above methods.
In still another aspect the invention provides
differentiated cells derived (directly or indirectly)
from the embryoid bodies.
In accordance with the present invention, primate ES

cells that have been cultured under standard conditions
(see e.g. U.S. patent 5,843,780) are permitted to
overgrow, pile up and/or otherwise closely associate in
clumps on a substrate (e.g. a plastic tissue culture
plate with standard feeder layer). They are then removed
as clumps from the substrate (e.g. by incubating the
colonies with an enzyme which attacks the ES cell
colony"s attachment to the substrate more strongly than
ES cell attachments with ES cells). In such a case the
enzyme could be dispase at a concentration of about 10
mg/ml.
Alternatively, the clumps could be removed as clumps
by mechanically scraping with a micropipette, cell
scraper, or the like.
The essentially intact colonies are then incubated
under non-attaching conditions (preferably continuous
rocking of the culture dish, culture on non-attaching
bacterial grade culture plastic, and/or continuous
culture in the presence of serum-free medium which lacks
serum attachment factors). The colonies can then quickly
coalesce into compact embryoid bodies, which can
thereafter be allowed to differentiate either in
continuous suspension, or after re-attachment to a
substrate. Such embryoid bodies can be used to derive
differentiated derivatives of endoderm, mesoderm, and
ectoderm, and for obtaining other desired lineages.
It is an advantage of the present invention that it
provides effective methods of forming primate embryoid
bodies from primate embryonic stem cell lines. Another
advantage of the present invention is to provide primate
embryoid bodies suitable for differentiation into other
primate cell types. Other features and advantages of the
present invention will become apparent after study of the
specification and claims which follow.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Formation Of Embryoid Bodies
Primate embryonic stem cells (e.g. rhesus or human -
U.S. patent 5,843,780; J. Thomson, et al., 282 Science
1145-1147 (1998)) are cultured on mitotically inactivated
(3000 rads g-radiation) mouse embryonic fibroblasts,
prepared at 5x104 cells/cm2 on tissue culture plastic
previously treated by overnight incubation with 0.1%
gelatin. E. Robertson, Embryo-derived Stem Cell Lines.
In: Teratocarcinomas And. Embryonic Stem Cells: A
Practical Approach IRL Press: Washington, D.C., 71-112
(1987). Culture medium consists of 79% Dulbecco"s
modified Eagle medium (DMEM; 4500 mg of glucose per
liter; without sodium pyruvate) , 20% fetal bovine serum
(FBS), 0.1 mM 2-mercaptoethanol, 1 mM L-glutamine and 1%
nonessential amino acid stock (GIBCO).
One allows colonies to form clumps over a period of
hours. ES cell colonies can then be removed from the
tissue culture plate using physical or chemical methods
that keep the ES cells in clumps.
For dispase or collagenase removal of ES cell
colonies from the culture plate, the culture medium is
removed from the ES cells. Dispase (10 mg/ml in ES
culture medium) or collagenase (1 mg/ml solution in DMEM
or other basal medium) is added to the culture plate. The
culture plates are returned to the incubator for 10-15
minutes.
After dispase treatment the colonies can either be
washed off the culture dishes or will become free of the
tissue culture plate with gentle agitation. After
collagenase treatment the cells can be scraped off the
culture dish with a 5 ml glass pipette. Some dissociation
of the colonies occurs, but this is not sufficient to
individualize the cells. After chemical removal of the
cells from the tissue culture plate, the cell suspension

is centrifuged gently for 5 minutes, the supernatant is
removed and discarded, the cells are rinsed, and the
cells are resuspended in culture medium with or without
serum.
Mechanical removal of the cells is achieved by using
a pulled glass pipette to scrape the cells from the
culture plate. Cell clumps can be immediately
resuspended, without centrifugation, in fresh tissue
culture medium.
Once colonies are removed from the tissue culture
plate, the ES cells should remain in suspension during
further embryoid body formation. This can be achieved
by, for example, gently and continuously rocking the cell
suspension. Cell suspensions are aliquoted into wells of
6-well tissue culture dishes, placed inside a sealed,
humidified isolation chamber, gassed with 5% CO2, 5%O2 and
90%N2 and placed on a rocker (Red Rocker, Hoefer
Scientific Instruments). The rocker is housed inside an
incubator maintained at 37°C. The culture plates can be
rocked continuously for at least 4 8 hours and up to 14
days.
Every 2 days the plates are removed from the rocking
device, the culture medium is removed, and fresh culture
medium is added to the cells. The culture dishes are then
returned to the rocking environment. Cells will also
remain in suspension when cultured in suspension culture
dishes (Nunc) without rocking, or when cultured in the
absence of serum, which provides attachment factors. All
cells must be cultured at 37°C, in a humidified,
controlled gas atmosphere (either 5% CO2, 5% O2 and 90% N2
or 5% CO2 in air).
Following culture in suspension for up to 11 days,
embryoid bodies are dispensed by mechanical or chemical
means and can be allowed to reattach to tissue culture
plates treated with gelatin or matrix, in ES medium.

Displaced, plated embryoid bodies will form flattened
monolayers and can be maintained by replacing medium
every 2 days.
Analysis Of Embryoid Bodies And Differentiated Cells
We used immunofluorescent antibody staining up to 7
days after plating to confirm the existence of cells of
the neural phenotype. Cells were fixed in 30%
methanol/10% acetic acid before incubation with
antibodies. Antibodies that were used are as follows:
rabbit anti-bovine GFAP (DAKO), anti-Forse-1
(Developmental Studies Hybridoma Bank) , anti-bovine MAP-2
(Roche), anti-human NCAM/CD56 (DAKO) and anti-01
(provided by S.-C. Zhang, University of Wisconsin). All
primary antibodies are mouse monoclonals except anti-
GFAP. Secondary antibodies, FITC-conjugated goat anti-
mouse IgG and biotin-conjugated goat anti-rabbit, as well
as AMCA-conjugated streptavidin were purchased from
Jackson ImmunoResearch.
The Forse-1 antibody recognizes phosphacan, a brain-
specific chondroitin sulfate proteoglycan that binds
neural cell adhesion molecules in the embryonic CNS of
both humans and rodents. K. Allendorfer et al. , 6 Mol.
And Cell. Neuro. 381-395 (1995); S. Tole et. al. , 15 J.
Neuro. 957-969 (1995). The 01 antibody identifies pro-
oligodendrocytes present from day 3 in embryonic mouse
brain cultures. M. Schachner et. al. , 83 Dev. Biol. 328-
338 (1981); I. Sommer, et al., 83 Dev. Biol. 311-327
(1981).
Within three days of plating, the neural precursors,
stained by Forse-1 and 01, were observed. The Forse-1
antibody stained numerous rounded cells, whereas very
sparse, flattened cells with extensive projections were
stained with the anti-01 antibody.
Neurons and glial were detected, 3 days and later
after plating, by positive staining of neural cell

adhesion molecule (NCAM)/CD56 (Figure 3), microtubule-
associated protein-2 (MAP-2) (Figure 3), ?III-tubulin and
glial fibrillary acidic protein (GFAP). NCAM is a cell
adhesion molecule thought to be important in cell-cell
interactions within the neuroepithelium. B. Cunningham,
et al., 236 Science 799-806 (1987); J. Ritz, et al., 42
Adv. Immuno. 181-211 (1988). MAP-2 plays an important
role in brain microtubule assembly. ?III-tubulin is a
neuron-specific marker, and glial fibrillary acidic
protein (GFAP) is an astrocyte marker.
Differentiation Into Lineages
Embryoid bodies can be differentiated into a variety
of desired lineages. For example the embryoid bodies
could be used to derive hematopoietic cells using
techniques analogous to those used for mouse in M. Wiles
et al. 111 Development 259-267 (1991). In this regard
one could plate the embryoid bodies in serum-containing
medium in the presence of 2 i.u./ml erythropoietin or IL-
3.
If cardiac lineages are desired one could use
techniques analogous to T. Doetschman et al., 87 J.
Embry. Exper. Morph. 27-45 (1985). One could plate the
bodies in serum-containing medium with no additives.
To develop neural lineages one could plate the
embryoid bodies in the presence of 20 ng/ml fibroblast
growth factor plus 20 ng/ml epidermal growth factor.
This is analogous to techniques described in B. Reynolds
et al., 255 Science 5052 (1992) .
The present invention thus provides an effective
method for making primate embryoid bodies from primate ES
cells. While the above work was focused on rhesus and
human embryonic stem cells (and neural cells derived
therefrom via these embryoid bodies), the techniques
described herein should work broadly for primate
embryonic stem cells and other cell types. Further,

while specific techniques for clump removal have been
discussed, the invention is not limited to those
alone. Rather, other techniques for removing the calls
in clumps from the substrate should work.
Thus the invention is not limited to the specific
embodiments described herein. Rather, the claim should
be looked to in order to judge the full scope of the
invention.
INDUSTRIAL APPLICABILITY
The present invention provides a supply of human
and other primate embryoid bodies suitable for
research, medical purposes, and differentiation into
lineages. The present invention does not encompass
reproductive cloning methodologies used to produce /
manufacture human or animals. The invention does,
however, include therapeutic (cellular) cloning useful
for drug discovery, toxicity screens, and
transplantation, which includes providing cells used
to treat for example, life-threatening cancers and
blood disorders, such as leukemia.

We claim:
1. A method for producing primate embryoid bodies
from colonies of primate embryonic stem cells that are
adhering to a substrate, the method comprising:
removing the adhering colonies of the embryonic stem
cells from the substrate in clumps; and
then incubating the clumps in a container under
conditions in which the clumps are essentially inhibited
from attaching to the container and coalesce into
embryoid bodies.
2. The method as claimed in claim 1, wherein the removal step
is conducted in the presence of an enzyme that promotes
disassociation of the clumps as clumps from the substrate.
3. The method as claimed in claim 2, wherein the enzyme is
dispase.
4. The method as claimed in claim 1, wherein the removal step
is conducted in the presence of a chelating agent.
5. The method as claimed in claim 1, wherein the removal step
comprises mechanically scraping the clumps from the
substrate.
6. The method as claimed in claim 1, wherein the removal step
is conducted in the presence of trypsin, calcium and
magnesium.
7. The method as claimed in claim 1, wherein the incubation
step comprises agitating the container.
8. The method as claimed in claim 1, wherein the incubation
step is conducted in a container made of plastic.
9. The method as claimed in claim 1, wherein the incubation
step is conducted in the presence of a serum-free medium.
10. The method as claimed in claim 1, wherein the primate
embryonic stem cells are human embryonic stem cells and
the primate embryoid bodies are human embryoid bodies.
Primate embryoid bodies are formed from primate ES cells. The ES cells form clumps. One then removes the
clumps, as clumps, and permits incubation under non-adherent conditions. The development of embryoid bodies from primate ES
cells is dependent on maintaining the aggregation of cells, as individualized cells will rapidly die.

Documents:

IN-PCT-2002-1133-KOL-CORRESPONDENCE 1.1.pdf

IN-PCT-2002-1133-KOL-CORRESPONDENCE 1.2.pdf

IN-PCT-2002-1133-KOL-CORRESPONDENCE.pdf

IN-PCT-2002-1133-KOL-FORM 15.pdf

IN-PCT-2002-1133-KOL-FORM-27.pdf

in-pct-2002-1133-kol-granted-abstract.pdf

in-pct-2002-1133-kol-granted-claims.pdf

in-pct-2002-1133-kol-granted-correspondence.pdf

in-pct-2002-1133-kol-granted-description (complete).pdf

in-pct-2002-1133-kol-granted-examination report.pdf

in-pct-2002-1133-kol-granted-form 1.pdf

in-pct-2002-1133-kol-granted-form 18.pdf

in-pct-2002-1133-kol-granted-form 2.pdf

in-pct-2002-1133-kol-granted-form 3.pdf

in-pct-2002-1133-kol-granted-form 5.pdf

in-pct-2002-1133-kol-granted-gpa.pdf

in-pct-2002-1133-kol-granted-letter patent.pdf

in-pct-2002-1133-kol-granted-reply to examination report.pdf

in-pct-2002-1133-kol-granted-specification.pdf

IN-PCT-2002-1133-KOL-OTHERS 1.1.pdf


Patent Number 213788
Indian Patent Application Number IN/PCT/2002/1133/KOL
PG Journal Number 03/2008
Publication Date 18-Jan-2008
Grant Date 16-Jan-2008
Date of Filing 05-Sep-2002
Name of Patentee WISCONSIN ALUMNI RESEARCH FOUNDATION
Applicant Address 614 WALNUT STREET, P.O.BOX 7365 MADISON, WI 53707-7365.
Inventors:
# Inventor's Name Inventor's Address
1 THOMSON JAMES A. 1807 REGENT STREET MADISON, WI 53705
2 MARSHALL VIVIENNE S 4326 BUCKEYE ROAD MADISON, WI 53716
3 SWIERGIEL JENNIFER J 10219 HAWKS POINT TRAIL, ROSCOE, IL 61073
PCT International Classification Number C12N 5/02
PCT International Application Number PCT/US01/05252
PCT International Filing date 2001-02-20
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 09/510,444 2000-02-21 U.S.A.