Note: Descriptions are shown in the official language in which they were submitted.
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
1
CELL DIFFERENTIATION/PROLIFERATION AND MAINTENANCE
FACTOR AND USES TI~iEREOF
The present invention relates to a biologically active factor, and more
particularly to a factor capable of influencing differentiation, proliferation
and/or
maintenance of pluripotent cells including embryonic stem (ES) cells. The
present
invention also relates to methods of using the biologically active factor to
produce
fram pluripotent cells, pluripotent cells having different properties, more
particularly EPL cells; and to methods of producing partially or terminally
differentiated cells from the piuripotent cells. The present invention also
relates to
7 0 pluripotent cells and partially or terminally differentiated cells, and
their uses.
initial developmental events within the mammalian embryo entail the
elaboration of extra-embryonic cell lineages and result in the formation of
the
blastocyst, which comprises trophectodeml, primitive endoderm and a pool of
pluripotent cells, the inner cell mass (ICM/epiblast). As development
continues,
the cells of the ICNUepiblast undergo rapid proliferation, selective
apoptosis,
differentiation and reorganisation as they develop to form the primitive
ectoderm.
In the mouse, the cells of the ICM begin to proliferate rapidly around the
time of
blastocyst implantation. The resulting pluripotent cell mass expands into the
blastocoelic cavity. Between 5.0 and 5.5 d.p.c. the inner cells of the
epiblast
undergo apoptosis to form the proamniotic cavity. The outer, surviving cells,
or
early primitive ectoderm, continue to proliferate and by 6.U-6.5 d.p.c. have
formed
a pseudo-stratified epithelial layer of pluripotent cells, termed the
primitive or
embryonic ectoderm. The primitive ectoderm gives rise to the germ cells and,
during gastrulation, acts as a substrate for the generation of the primary
germ
layers of the embryo proper and the extra-embryonic mesodeml.
The analysis of developmental potential and differentially expressed genes
of temporally distinct pools of pluripotent cells within the pre-gastrulation
embryo
has identified two distinct populations of pluripotent cells, the 1CM and
primitive
ectoderm. Cells within each population appear homogenous as revealed by
transplantation studies and the analysis of gene expression markers. The
establishment of pluripotent cell heterogeneity and the determination of sub-
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
2
populations of pluripotent cells within the primitive ectoderm, as defined by
differential gene expression, has not been documented in embryos prior to
formation of the primitive streak at 6.5 d.p.c. A paucity of genetic markers
and the
small size, complexity and relative inaccessibility of the embryo within the
uterine
environment between 4.5 and 6.5 d.p.c. has not allowed a comprehensive
analysis of pturipotent cell progression.
Pluripotent cells can be isolated from the pretmplantation mouse embryo as
embryonic stem (ES) cells. ES cells can be maintained indefinitely as a
pluripotent cell population in vitro, and, when reintroduced into a host
blastocyst,
can contribute to all adult tissues of the mouse including the germ cells. ES
cells,
therefore, retain the ability to respond to all the signals that regulate
normal mouse
development, and potentially represent a powerful model system for the
investigation of mechanisms underlying pluripotent cell biology and
differentiation
within the early embryo, as well as providing opportunities for embryo
manipulation and resultant commercial, medical and agricultural applications.
Other pluripotent cells and cell fines will share some or all of these
properties and
applications.
The successful isolation, long term clonal maintenance, genetic
manipulation and germ-line transmission of pluripotent cells from species
other
than rodents has generally been difficult to date and the reasons for this are
unknown. lntemational patent application W097I32033 and US Patent 5,453,357
describe pturipotent cells including cells from species other than rodents,
and
primate pluripotent cells have been described in International patent
applications
W098/43679 and W096/23362 and in US Patent 5,843,780. However, it would
be useful if these pluripotent cells could be transformed into pluripotent
cells with
different properties.
The differentiation of ES cells can be regulated in vitro by the cytokine
leukaemia inhibitory factor (L1F) and other gp130 agonists which promote self-
renewal and prevent differentiation of the stem cells. However, with the
exception
of retinoic acid, biological molecules that can induce the differentiation of
ES cells
CA 02324591 2000-09-19
WO 99/33021 PCT/AU99/00265
3
into specific cell types, in the presence or absence of LIF, are currently
unknown.
It is an object of the present invention to overcome, or at least alleviate,
one
or more of the difficulties or deficiencies associated with the prior art.
Applicant has identified a biologically active factor which is capable of
influencing differentiation, proliferation and/or maintenance of pluripotent
cells
including ES cells. More specifically, the factor is capable of causing the
transition
of pluripotent cells (e.g. ES cells in adherant culture and in suspension
culture) to
pluripotent cells having different properties, more specifically early
primitive
ectodem~-like {EPL) cells. Additionally the factor is capable of maintaining
and
supporting proliferation of these cells in vifro. tt also allows the isolation
and
maintenance of EPL cells derived from in vitro and in vivo primitive ectoderm.
Whilst applicant does not wish to be restricted by theory, it is thought that
EPL cells represent an in vitro equivalent of the pluripotent cells of a post
implantation embryo prior to fi.0 d.p.c. or early primitive ectoderm.
Accordingly, it should be understood that the term "EPL cells" refers to cells
derived from piuripotent cells that retain pluripotency and are converted to
and/or
maintained as cells that express Oct4 and FgfS by:
{a) the biologically active factor of the present invention or the large or
low
molecular weight component thereof; or
(b) a conditioned medium according to the present invention; or
(c) an extracellular matrix in the presence or absence of the tow molecular
weight component according to the present invention.
The pluripotent cells from which the EPL cells may be derived may be but
are not restricted to ES cells, in vitro or in vivo derived ICM/epiblast, in
vitro or in
vivo derived primitive ectoderm, teratocarcinoma cells, EC cells, primordial
germ
cells, EG cells and pluripotent cells derived by dedifferentiation or by
nuclear
CA 02324591 2000-09-19
WO 99153021 PCT/AU99100265
4 -
transfer. EPL cells may also be derived from differentiated cells by
dedifferentiation.
Furthermore, the EPL cells may be capable of but are not restricted to
reversion to ES cells by the removal of (a), {b) or (c) in the presence of a
gp130
agonist.
The biologically active factor preferably includes two components, a low
molecular weight component and a large molecutar weight component.
The low molecular weight component may be an amino acid or functionally
active analogue thereof or a peptide or functionally active fragment or
analogue
thereof. Preferably the low molecular weight component is proline or a
functionally active analogue thereof or a peptide including proline or a
functionally
active fragment or analogue thereof. Even more preferably the low molecular
weight component is L-proline or a peptide including L-proline. The peptide
preferably has a molecular weight of less than approximately 5 kD, more
preferably less than approximately 3 kD. Alternatively or in addition, the
peptide
preferabiy has a size of approximately 1-11, more preferably approximately 1-
7,
most preferably approximately 1-4 amino acids. Most preferably the peptide is
selected from the group consisting of:
Pro-ala
Ala-pro
Ala-pro-gly
Pro-OH-pro
Pro-gly
Gly-pro
Gly-pro-ala
Gly-pro-OH-pro
Giy-pro-arg-pro
Gly-pro-gly-gly
Val-ala-pro-gly
Substance P frag. 1-4 (Arg-pro-lys-pro)
Substance P free acid
CA 02324591 2000-09-19
WO 99153021 PCTIAU99/00265
(arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-gly-Leu-metOH)
Protease digested (including cotlagenase digested) collagen fragments;
and functionally active fragments and analogues thereof; and molecules
which compete therewith for biological activity.
By the term "functionally active" is meant that the fragment or analogue is
capable of influencing differentiation, proliferation andlor maintenance of
pluripotent cells including ES cells.
The large molecular weight component may be a polypeptide or protein.
Preferably the large molecular weight component has a molecular weight of
greater than approximately 10 kD, more preferably between approximately
50-1000 kD, most preferably between approximately 100-500 kD. The large
molecular weight component may be in solution or contained in an extracellular
matrix.
In a preferred form of this aspect of the invention the large molecular weight
component may be an extracellular matrix protein or functionally active
fragment
ar analogue thereof, for example a fibronectin or laminin or functionally
active
fragment or analogue thereof. In a particularly preferred form the large
molecular
weight component is a cellular fibronectin. The cellular fibronectin may have
a
molecular weight of approximately 210 to 250 kD, as measured on a 10%
reducing/denaturing polyacrylamide gel.
In a further aspect of the present invention there is provided a composition
capable of influencing differentiation, proliferation andlor maintenance of
pluripotent cells including ES cells, said composition including a
biologically active
factor and/or a low molecular weight component andlor a large molecular weight
component as hereinbefore described, together with an adjuvant, diluent or
carrier.
The biologically active factor may be isolated from a medium conditioned by
cultured cells. Alternatively, the components of the biologically active
factor may
be purified from other sources, synthesised or recombinantly produced.
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99J00265
Accordingly, in a further aspect of the present invention there is provided a
conditioned medium capable of influencing differentiation, proliferation
and/or
maintenance of pluripotent cells, or a fraction thereof including medium
components below approximately 5 kDa, and/or a fraction thereof including
medium components above approximately 10 kDa.
Preferably the conditioned medium includes a biologically active factor as
hereinbefore described or the low or large rnolecutar weight component
thereof.
Preferably the conditioned medium is prepared by a method as hereinafter
described.
Preferably the conditioned medium is prepared using a hepatic or
hepatoma cell or cell line, more preferably a human hepatocellular carcinoma
cell
line such as Hep G2 cells (ATCC HB-8065) or Hepa-1 c1 c-7 cells (ATCC CRL-
2026), primary embryonic mouse liver cells, primary adult mouse liver cells,
or
primary chicken fiver cells, or an extraembryonic endodermal cell or cell line
such
as the cell lines END-2 and PYS-2. However, the biologically active factor may
be
isolated from a medium conditioned by liver or other cells from any
appropriate
species, preferably mammalian or avian. Alternatively, the activity may also
be
derived by contributions from two or more different conditioned media from
cells
which express one or the other of the components.
Accordingly, in a further aspect of the present invention, there is provided a
method for preparing a conditioned medium capable of influencing
differentiation,
proliferation and/or maintenance of pluripotent cells, said method including
providing
cells, and
a cell culture medium;
culturing the cells in the cell culture medium for a time sufficient to
produce
the conditioned medium; and
separating the cells from the culture medium to provide the conditioned
medium.
CA 02324591 2000-09-19
WO 99153021 PCTlAU99100265
7 _
The cells may be of any suitable type. Preferably the cells are hepatic or
hepatoma cells or a hepatic or hepatoma cell line. More preferably they are
selected from the group consisting of a human hepatocellular carcinoma cell
fine,
primary embryonic mouse liver cells, primary adult mouse liver cells and
primary
chicken liver cells. Most preferably a human hepatocellular carcinoma cell
fine
such as Hep G2 cells (ATCC HB-8065) or Hepa-1 c-1 c-7 cells (ATCC CRL-2026)
is used. Alternatively the cells may be endodermal cells, more preferably
primary
or cultured extraembryonic cells or cell Lines such as the endodermal cell
Lines
END-2 and PYS-2.
The cells may be cultured under conditions suitable for their proliferation
and maintenance in vitro. This includes the use of serum including fetal calf
serum and bovine serum or the medium may be serum-free. Other growth
enhancing components such as insulin, transferrin and sodium selenite rnay be
added to improve growth of the cells from which the conditioned medium or
extracellular matrix rnay be derived. As would be readily apparent to a person
skilled in the art, the growth enhancing components will be dependent upon the
cell types cultured, other growth factors present, attachment factors and
amounts
of serum present.
The cel~s may be cultured for a time sufficient to establish the cells in
culture. By this we mean a time when the cells equilibrate in the culture
medium.
Preferably the cells are cultured for approximately 3-5 days.
The cell culture medium may be any cell cuEture medium appropriate to
sustain the cells employed. Where the cells are a fiver cell or liver cell
fine, the
culture medium is preferably DMEM containing high glucose, supplemented with
10% FCS, 40 p.glml gentamycin, 1 mM L-glutamine, 37°C, 5% C02.
Separation of the cell culture medium from the cells may be achieved by
any suitable technique, such as decanting the medium from the cells.
Preferably
the cell culture medium is clarified by centrifugation or filtration (e.g.
through a
0.22 ~.M filter) to remove excess cells and cellular debris. Other known means
of
separating the cells from the medium may be employed providing the separation
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
8 _
method does not remove the growth components from the medium.
In a still further aspect of the present invention there is provided a
substantially or partially purified extracelluiar matrix capable of
influencing
differentiation, proliferation and/or maintenance of piuripotent cells.
Preferably the extracellular matrix includes a biologically active factor as
hereinbefore described or the large molecular weight component thereof.
Preferably the extracellular matrix is prepared by a method as hereinafter
described.
Accordingly, in a further aspect of the present invention there is provided a
method for preparing an extracellular matrix capable of influencing
differentiation,
proliferation, and/or maintenance of pluripotent cells, said method including
providing
cells,
a support substrate, and
a cell culture medium;
culturing the cells in the cell cutture medium in the presence of the support
substrate for a time sufficient to produce the extracelluiar matrix; and
separating the cells and the culture medium from the support substrate to
provide the extraceliular matrix.
The cells may be of any suitable type. Preferably the cells are hepatic or
hepatoma cells or a hepatic or hepatoma cell line. More preferably they are
selected from the group consisting of a human hepatocetlular carcinoma cell
fine,
primary embryonic mouse liver cells, primary adult mouse fiver cells and
primary
chicken liver cells. Most preferably a human hepatoceliular carcinoma cell
line
such as Hep G2 cells (ATCC HB-8065) or Hepa-1 c-1 c-7 cells (ATCC CRL-2026)
is used. Alternatively the cells may be endodermal cells, more preferably
primary
or cultured extraembryonic cells or cell lines such as the endodermal cell
lines
END-2 and PYS-2.
CA 02324591 2000-09-19
WO 99/53021 PCTIAU99/002b5
g _
The cells may be cultured as in accordance with the conditions described
for preparation of conditioned medium. Isolation of the matrix-associated
activity
involves separation of the cell culture medium and appropriate cells or cell
lines
from the ECM and support substratum. This may involve washing the matrix with
a suitable buffer. Preferably cells are cultured in 0.2% azide, 0.1 mM PMSF in
phosphate buffered saline {PBS) for fi-18 hours to kill and detach cells or
cultured
in 0.5 mM EGTA for 15 minutes to detach cells, with cells and debris removed
by
further washes with PBS.
Alternatively, an extracellular matrix capable of influencing differentiation,
proliferation and/or maintenance of pfuripotent cells may be prepared from
animal
sources such as visceral, parietal or primitive endoderm, or from animal cells
and
tissues, e.g. placenta. An extracellular matrix rnay also be prepared by
artificial
means such as drying down purified or semi purified extracellular matrix
components on tissue culture plates.
Applicant has found that biologically active factors prepared using a human
hepatocellular carcinoma cell line, primary embryonic mouse liver cells,
primary
adult mouse liver cells and primary chicken Liver cells are capable of
influencing
differentiation, proliferation andlor maintenance of mouse ES cells. Thus,
whilst
applicant does not wish to be restricted by theory, it appears that the factor
is
biologically active across species.
The components of the biologically active factor may be partially or
substantially purified. Purification of the components may be carried out by
removal of the cells followed by techniques such as cation exchange
chromatography, hydrophobic interaction chromatography, anion exchange
chromatography, heparin affinity chromatography, size exclusion
chromatography,
ultrafiltration, normal phase chromatography andlor reverse phase
chromatography. Preferably, purification of the components is carried out
using
FPLC and HPLC chromatographic techniques.
In a preferred form of this aspect of the invention, the low molecular weight
component may be purified by a combination of optional fractionation eg.
CA 02324591 2000-09-19
WO 9915302! PCTIAU99100265
-
ultrafiltration, gel filtration chromatography (preferably on a Sepharose G10
column), normal phase chromatography and gel filtration chromatography
(preferably on a Superdex peptide gel filtration column}; and preferably
sequentially in the stated order.
5 Accordingly, the present invention provides a method for partially or
substantially purifying a low molecular weight component of a biologically
active
factor capable of influencing differentiation, proliferation and/or
maintenance of
pluripotent cells, said method including
providing
10 a source of said low molecular weight component,
a first gel filtration chromatography support,
a normal phase chromatography support, and
a second gel filtration chromatography support;
contacting the source of said low molecular weight component with the first
gel filtration chromatography support to produce a first fraction;
contacting said first fraction with the normal phase chromatography support
to produce a second fraction; and
contacting said second fraction with the second gel filtration
chromatography support to produce the partially or substantially purified low
molecular weight component.
Preferably, the method includes the further step of subjecting the source of
said low molecular weight component to a fractionation step, e.g.
ultrafiltration to
obtain a fraction containing components with a molecular weight of less than
approximately 3 kD.
in a further preferred form of this aspect of the invention, the large
molecular weight component may be purified by a combination of affinity
chromatography (preferably heparin sepharose affinity chromatography, e.g on a
heparin sepharose CL-6B column), anion exchange chromatography (e.g. using a
Resource Q anion exchange column} and gel filtration chromatography
(preferably
on a Superose 6 gel filtration column); and preferably sequentially in the
stated
order.
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
11 -
Accordingly, the present invention provides a method for partially or
substantially purifying a large molecular weight component of a biologically
active
factor capable of influencing differentiation, proliferation and/or
maintenance of
piuripotent cells, said method including
providing
a source of said large molecular weight component,
a heparin affinity chromatography support,
an anion exchange chromatography support, and
a gel filtration chromatography support;
contacting the source of said large molecular weight component with the
heparin affinity chromatography support to produce a first fraction;
contacting said first fraction with the anion exchange chromatography
support to produce a second fraction; and
contacting said second fraction with the gel filtration chromatography
support to produce the partially or substantially purified .large molecular
weight
component.
Alternatively, the large molecular weight component may be purified by a
combination of optional fractionation eg uitrafiltration (e.g. using a Amicon
DiaFlo
YM10 membrane), anion exchange chromatography (e.g. using a Sepharose Q
anion exchange column), hydrophobic interaction chromatography (e.g. using a
phenyl sepharose hydrophobic interaction column), heparin affinity
chromatography (.e.g. heparin sepharose affinity chromatography eg. using a
heparin sepharose CL-6B column) and get filtration (e.g. using a Superose 6
gel
filtration column); and preferably sequentially in the stated order.
Accordingly, the present invention provides a method for partially or
substantially purifying a large molecular weight component of a biologically
active
factor capable of influencing differentiation, proliferation andlor
maintenance of
pluripotent cetis, said method including
providing
a source of said large molecular weight component,
an anion exchange chromatography support,
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
12 -
a hydrophobic interaction chromatography support,
a heparin affinity chromatography support, and
a gel filtration chromatography support;
contacting the source of said large molecular weight component with the
anion exchange chromatography support to produce a first fraction;
contacting said first fraction with the hydrophobic interaction
chromatography support to produce a second fraction;
contacting said second fraction with the heparin affinity chromatography
support to produce a third fraction; and
contacting said third fraction with the gel filtration chromatography support
to produce the partially or substantially purified large molecular weight
component.
Preferably, the method includes the further step of subjecting the source of
said high molecular weight component to a fractionation step, e.g,
ultrafiltration to
obtain a fraction containing components with a molecular weight of greater
than
approximately 10 kD.
Alternatively the large molecular weight component may be purified by
gelatin affinity chromatography, preferably gelatin sepharose affinity
chromatography, optionally followed by dialysis.
Accordingly, the present invention provides a method for partially or
substantially purifying a large molecular weight component of a biologically
active
factor capable of influencing differentiation, proliferation and/or
maintenance of
pluripotent cells, said method including
providing
a source of said large molecular weight component,
a gelatin afi~nity chromatography support, and
a dialysis system;
contacting the source of said large molecular weight component with the
gelatin affinity chromatography support to produce a first fraction; and
subjecting said first fraction to dialysis to produce the partially or
substantially purified large molecular weight component.
CA 02324591 2000-09-19
WO 99153021 PCTIAU99100265
13
Preferably, the methods for purification of the large molecular weight
component include the further step of subjecting the source of said large
molecular weight component to a fractionation step, e.g. ultrafiltration to
obtain a
fraction containing components with a molecular weight of greater than
approximately 10 kD. This step may be included at any appropriate point in the
purification procedure.
Preferably the source of the large or low molecular weight component is a
partially purified biologically active factor as hereinbefore described, a
conditioned
medium as hereinbefore described or an extracellular matrix as hereinbefore
described.
A conditioned medium as hereinbefore described may be used to derive
and maintain pluripotent cells including EPL cells or the medium may be
fractionated fo yield the biologically active factor or components thereof,
which
may be added alone or in combination to other media to provide a pluripotent
cell,
18 e.g. EPL cell deriving and maintaining medium. Alternatively, partially or
substantially purified or synthetic or recombinent forms of the biologically
active
factor or components thereof may be added to other media alone or in
combination to provide a pluripotent cell, (e.g. EPL cell) deriving and
maintaining
medium. The conditioned medium may be used undiluted or diluted (e.g. approx.
i 0-80%).
When the low molecular weight component is proline, its concentration in
the cel! maintaining medium is preferably in the range of approximately 40 ~cM
or
greater. When the large molecular weight component is cellular fibronectin its
concentration in the cell maintaining medium is preferably in the range of
approximately 2 p.g/ml or greater. Extracellular matrix from relevant cells
and cell
lines or extracellular matrix prepared artificially may also be used to
provide the
large molecular weight component of the biological activity. The conditioned
medium, factor or large molecular weight component thereof may also be used to
coat culture plates to provide the desired biological activity.
The biologically active factor of the present invention or the high or low
CA 02324591 2000-09-19
WO 99/53021 PC'T/AU99/002G5
14 -
molecular weight components thereof may be used to produce pturipotent cells
including EPL cells, not only from rodents, but also from other more
commercially
important species including humans.
Accordingly, in a further aspect of the present invention there is provided a
method of producing and/or maintaining early primitive ectoderm-like (EPL)
cells,
said method including
providing
pluripotent cells , and
a biologically active factor according to the present invention, or the
large or low molecular weight component thereof; or
a conditioned medium according to the present invention, or
an extraceliular matrix according to the present invention and
optionally the low molecular weight component of the biologically active
factor of the present invention; and
contacting the pluripotent cells with the biologically active factor or the
large
or low molecular weight component thereof, or the conditioned medium, or the
extracellular matrix, to produce or maintain the EPL cells.
The pluripotent cells may be selected from the group consisting of
embryonic stern (ES) cells, in vivo or in vitro derived ICM/epibiast, in vivo
or in
vitro derived primitive ectoderm, primordial germ cells, EG cells,
teratocarcinoma
cells, EC cells, and piuripotent cells derived by dedifferentiation or by
nuclear
transfer. EPL cells may also be derived from differentiated cells by
dedifferentiation.
The biologically active factor or the large or low molecular weight
components thereof or the conditioned medium or the extracellular mat~uc
optionally plus low molecular weight component may be used for the isolation,
proliferation or maintenance of EPL cells in vitro. EPL cells may be generated
in
adherent culture or as cell aggregates in suspension culture. The biologically
active factor or components of the biologically active factor or the
conditioned
medium or the extracellular matrix optionally plus low molecular weight
component
may also be used alone or in combination with other factors to generate
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99100265
15 -
differentiated cells, tissues or organs by techniques known to those skilled
in the
art. The differentiated cells derived from EPL cells may be used in alto,
concordant or xenotransplantation, cell therapy, tissue and organ augmentation
or
replacement, and gene therapy.
In one form of this aspect of the invention, the piuripotent cells may be
contacted with the biologically active factor or the large or low molecular
weight
component thereof or the conditioned medium or the extracellular matrix
optionally
plus low molecular weight component in the presence of a gp130 agonist such as
the cytokine leukaemia inhibitory factor (LIF) preferably at a concentration
of
greater than approximately 100 units/ml and more preferably greater than
approximately 1000 units/ml. Oncostatin M, CNTF, CT1 or IL6 with the soluble
IL6 receptor, and IL11 and other gp130 agonists at equivalent levels may also
be
used.
In the mouse the pluripotent cells may be ES cells or cells derived from
pluripotent cells of ICM/epiblast of embryos or cellular aggregates (embryoid
bodies) or primitive ectoderm derived from either embryos or from
differentiation in
vitro of ES cells as embryoid bodies or cellular aggregates. In other species
pluripotent cells may be derived from equivalent cell sources at the stages
relevant to each species. The source of pluripotent cells from all species may
include cells derived from primordial germ cells or teratocarcinomas. In
addition
pluripotent cells may be derived by dedifferentiation (e.g. by reverting
differentiated cells to a pluripotent state), or by application of nuclear
transfer
techniques, (e.g. when the nucleus of a differentiated or partially
differentiated cell
is transferred into an oocyte or early embryonic cell). The pluripotent cells
may be
from any vertebrae including murine, human, bovine, ovine, porcine, caprine,
equine and chicken. The cells may be isolated by any method known to the
skilled addressee.
in a preferred form of this aspect of the invention, the method includes the
further step of:
identifying the EPL cells.
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
16
The conversion of pluripotent cells to EPL cells may be assessed by
expression of marker genes (RNA transcripts and cell surface markers), cell
morphology, cytokine responsiveness and/or by differentiation in vitro or in
vivo.
Marker genes which may be used to assess the conversion of pluripotent
cells to EPL cells include known markers such as Rext, FgfS, Oct4 alkaline
phosphatase, uvomonriin, AFP, H19, Evxl, brachyury, and novel marker genes,
identified by the inventors, such as L17, Psc1 and K7. Marker genes down
regulated during transition from ES cells to EPL cells include Rexl, L17 and
Psc7.
FgfS and K7 are up regulated during this transition. Pluripotent cell markers
Oct4,
Alkaline phosphatase and uvomorulin are expressed by both ES cells and EPL
cells in similar levels. Other genes that are expressed in partially
differentiated or
differentiated embryonic or extraembryonic lineages such as AFP, H19, Evx1 and
brachyury are not expressed in any ES or EPL cells.
Cytokines and growth factors which act differently on EPL and other
pluripotent cells include members of the FGF family (e.g. aFGF, bFGF and FGF4)
which induce differentiation of EPL cells but not ES cells, and the TGFji
family,
(e.g. Activin A) which induces differentiation of EPL but not ES cells. In
addition
EPL cells are maintained in culture by levels of LIF approximately 10 fold
lower
than required for ES cell maintenance.
EPL cell differentiation can be distinguished from that of other pluripotent
cells by the rates and proportions of structures and differentiated cell types
such
as primitive ectoderm, visceral endoderm, nascent mesoderm, cardiac muscle,
macrophages and neurons formed within cell aggregates or embryoid bodies.
When compared to ES cells EPL cells undergo accelerated formation of late
primitive ectoderm, accelerated and increased formation of nascent mesoderm,
beating cardiocytes and macrophages, and decreased or absent formation of
visceral endoderm and neurons. EPL cells do not contribute to embryo
development following blastocyst injection, whereas ES cells and EG cells do.
EPL cells do not contribute to embryo development following blastocyst
injection,
whereas ES cells and EG cells do.
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
17 -
The EPL cells may be maintained in a pluripotent state by culture in the
presence of the biologically active factor or components thereof or the
conditioned
medium or the extracellular matrix optionally plus low molecular weight
component, in the presence or absence of additional gp130 agonist until
further
differentiation, induced by factors, conditions or procedures is initiated.
Accordingly in a further aspect of the present invention there is provided a
method of producing partially differentiated andlor terminally differentiated
cells,
said method including
providing
pluripotent cells, and
a biologically active factor according to the present invention, or the
large or low molecular weight component thereof, or
a conditioned medium according to the present invention, or
an extracellular matrix according to the present invention and
optionally the low molecular weight component of the biologically active
factor of the present invention;
contacting the pluripotent cells with the biologically active factor or the
large
or low molecular weight component thereof, or the conditioned medium, or the
extracellular matrix, in the presence or absence of additional gp130 agonist,
to
produce early primitive ectoderm-like (EPL) cells; and
manipulating the environment of the EPL cells to produce the partially
differentiated andlor terminally differentiated cells.
The pluripotent cells may be selected from the group consisting of
embryonic stem (ES) cells, in vivo or in vitro derived lCM/epiblast, in vivo
or in vitro
derived primitive ectoderm, primordial germ cells, EG cells, teratocarcinoma
cells,
EC cells, and pluripotent cells derived by dedifferentiation or by nuclear
transfer.
EPL cells may also be derived from differentiated cells by dedifferentiation.
In a preferred form of this aspect of the invention the environment of the
EPL cells may be manipulated by maintenance or removal of the biologically
active factor or components thereof or the conditioned medium or the
extracellular
matrix, in the presence or absence of one or more differentiation agents, e.g.
CA 02324591 2000-09-19
WO 99153021 PGT/AU99I00265
18
growth factors. For example, maintaining contact of EPL cells with the
conditioned medium, biologically active factor, or components thereof,
preferably
in cell aggregates grown in suspension, preferably in the presence of a growth
factor from the FGF family such as FGF4, may be used to produce an at least
partially differentiated cell type equivalent to embryonic neurectoderm, which
can
differentiate further to a range of neural cell types. The neurectoderm is
derived
via a cell type equivalent to embryonic ectoderm, which can differentiate
further to
a range of other ectodermal cell types.
tn the absence of the biologically active factor, or components thereof, and
preferably in the presence of a member of the FGF family, such as FGF4, a
growth factor from the TGF-~i family may be added to EPL cells to produce an
at
least partially differentiated cell type equivalent to embryonic nascent
mesoderm,
which can di#ferentiate further to a range of mesodermal cell types.
In a further preferred form of this aspect of the invention embryoid bodies or
aggregates formed from pluripotent cells in the presence of conditioned
medium,
or biologically active factor, or components thereof, may be disaggregated to
a
single cell suspension. When reaggregated in the absence of conditioned
medium, or biologically active factor, or components thereof, preferably in
the
presence of a member of the FGF family leg FGF4) these cells differentiate
predominantly to an at least partially differentiated cell type equivalent to
embryonic nascent mesoderm, which can differentiate further to a range of
mesodermal cell types including blood and muscle lineages.
The pluripotent cetls may be selected from the group consisting of
embryonic stem (ES) cells, in vivo or in vitro derived ICM/epiblast, in vivo
or in vitro
derived primitive ectoderm, primordial germ cells, EG cells, teratocarcinoma
cells,
EC cells, and pluripotent cells derived by dedifferentiation or by nuclear
transfer.
EPL cells may also be derived from differentiated cells by dedifferentiation.
in a further preferred form of this aspect of the invention EPL cell
differentiation is achieved by a process of first forming monolayer cultures
of EPL
cells, and then further culturing the cells in the presence of members of the
FGF
CA 02324591 2000-09-19
WO 99/53021 PC'fIAU99100265
19 -
family or TGF(3 family. Cells derived by this process are differentiated cells
with
distinctive morphologies.
Furthermore, formation of EPL cell aggregates in suspension culture, in the
absence of conditioned medium, or biologically active factor, or components
thereof, by aggregation of EPL cells formed in adherent or suspension culture,
results in the differentiation of these cells in the absence of cell types,
such as
visceral endoderm, which are known to influence the differentiation of
pluripotent
cells in the embryo. Aggregated EPL cells differentiated in this manner form
high
levels of nascent mesoderm that can be further differentiated to form beating
muscle and blood and other tissues of mesodermal origin.
In a further preferred form of this aspect of the invention, the method
includes the further step of:
identifying the partially or terminally differentiated cells by procedures
including cell surface markers and gene expression markers, morphology and
differentiation potential.
Marker genes which may be used to assess the conversion of pluripotent
cells to neurectoderm cells and neural lineages include known markers such as
Gbx2, Sox7, Sox2, nestin, N-Cam, Ocf4, FgfS and brachyury. Markers down
regulated during the transition from piuripotent cells to neurectoderm include
Oct4
and FgfS. Markers up regulated during this transition include Gbx2, Soxl,
Sox2,
nestin and N-Cam. Markers not expressed during this transition include
brachyury.
Marker genes which may be used to assess the conversion of pluripotent
cells to mesoderm and derivatives include known markers such as Ocf4, Fgf5 and
brachyury and Nkx 2.5 Markers down regulated during the transition from
pluripotent cells to mesoderm include Oct4 and FgfS. Markers up regulated
during
this transition include brachyury and, as the mesoderm differentiates into
muscle
precursors, Nkx 2.5.
Mesoderm and ectoderm germ layer cells can be distinguished on the basis
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
of their differentiation potential in vitro. Mesoderm germ layer can
differentiate
into muscle and blood lineages but not neurectoderm or neural lineages.
Ectoderm germ layer can differentiate into neurectoderm and neural iineages
but
not muscle and blood lineages.
5 In a further aspect of the present invention there is provided a method for
producing predominantly ectodermal germ layer cells, for producing from them
predominantly partially differentiated ectodermal and neurectodermal cells,
and
also for producing predominantly terminally differentiated ectodem~al cells,
including but not restricted to dermal cells, and also for producing
predominantly
10 terminally differentiated neuronal cells.
There is also provided a method for producing predominantly mesodermal
germ layer cells, for producing from them predominantly partially
differentiated
mesodermal cells, and also for producing predominantly terminally
differentiated
mesodem~al cells, including but not restricted to blood cells and beating
15 cardiocytes.
In a further aspect of the present invention there is provided an ectodermal
germ layer cell, a neurectodermai cell, a partially differentiated ectodermai
cell or
partially differentiated neurectodermal cell, and a terminally differentiated
ectodermal cell, such as a dermal cell, and a terminally differentiated
neuronal cell
20 produced by the method of the present invention.
In a further aspect of the present invention there is provided a mesodermal
germ layer cell, a partially differentiated mesodermal cell, and a terminally
differentiated mesodermal cell, such as a blood cell and muscle cell produced
by
the method of the present invention.
In a further aspect of the present invention there is provided a cultured EPL
cell.
Alternatively, the EPL cells may be reverted to ES cells by removal of the
conditioned medium, or biologically active factor, or components thereof in
the
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
21
presence of a gp130 agonist such as LIF preferably at levels of about 1000
units/ml or greater or other gp130 agonists at equivalent levels. This is
accompanied by reestablishment of ES cell gene expression, cytokine
responsiveness, morphology and differentiation potential, both in vivo and in
vitro.
This provides means for generating ES cells by reversion or dedifferentiation
of
primitive ectoderm or EPL cells from any desired species, and particularly
from
species where ES cells have not been available previously.
Accordingly, in a further aspect of the present invention, there is provided a
method of producing ES cells, said method including
providing
pfuripotent cells,
a biologically active factor or large or low molecular weight
component thereof according to the present invention, or
a conditioned medium according to the invention, or
an extracellular matrix according to the invention and optionally the
low molecular weight component of the biologically active factor according
to the present invention; and
a gp130 agonist;
contacting the pluripotent cells with the biologically active factor or the
large
or low molecular weight component thereof, or the conditioned medium, or the
extracellular matrix; and in the presence or absence of additional gp130
agonist to
produce EPL cells; and
contacting the EPL ceEls with the gp130 agonist in the absence of the
biologically active factor or the large or low molecular weight component
thereof,
or the conditioned medium, or the extracellular matrix; to enable the EPL
cells to
revert to ES cells.
The pluripotent cells may be of any suitable type, as hereinbefore
described.
The method may include the further step of differentiating the ES cells to
produce partially ar terminally differentiated cells as hereinbefore
described.
CA 02324591 2000-09-19
WO 99!53021 PCT/AU99/00265
22 -
Preferably the gp130 agonist is leukaemia inhibitory factor (LIF). However,
other gp130 agonists such as Oncostatin M, CNTF, CT1, or IL6 with soluble IL6
receptor at levels required for maintaining pluripotent ES cells in vitro, may
also be
used.
in a further aspect of the present invention, there is provided a method of
producing genetically modified ES cells, said method including:
providing
pluripotent cells,
a biologically active factor or large or low molecular weight
component thereof according to the present invention, or
a conditioned medium according to the invention, or
an extraceliular matrix according to the invention and optionally the
low molecular weight component of the biologically active factor according
to the present invention; and
i 5 a gp130 agonist;
contacting the pluripotent cells with the biologically active factor or the
large
or low mofecutar weight component thereof, or the conditioned medium, or the
extracellular matrix in the presence or absence of additional gp130 agonist to
produce EPL cells;
modifying one or more genes in the EPL cells; and
contacting the genetically modified EPL cells with the gp130 agonist in the
absence of the biologically active factor or the large or low molecular weight
component thereof, or the conditioned medium or the extracellular matrix; to
enable the genetically modified EPL cells to revert to genetically modified ES
cells.
The pluripotent cells may be of any suitable type, as hereinbefore
described.
It should be noted that genetically modified ES cells may also be produced
according to this method, where the reverted ES cells, and not the EPL cells,
are
genetically modified.
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
23
The method may include the further step of differentiating the genetically
modified ES cells to produce genetically modified partially or terminally
differentiated cells by methods as hereinbefore described including formation
of
EPL cells.
Alternatively, genetically modified partially or terminally differentiated
cells
may be produced by a method which includes
providing
pluripotent cells,
a biologically active factor or large or low molecular weight
component thereof according to the present invention, or
a conditioned medium according to the invention, or
an extracellular matrix according to the invention and optionally the
!ow molecular weight component of the biologically active factor according
to the present invention; and
a gp130 agonist;
contacting the piuripotent cells with the biologically active factor or the
large
or low molecular weight component thereof, or the conditioned medium, or the
extraceliular matrix; in the presence or absence of additional gp130 agonist
to
produce EPL cells;
modifying one or more genes in the EPL cells; and
differentiating the genetically modified EPL cells to produce the genetically
modified partially or terminally differentiated cells.
Alternatively partially or terminally differentiated cells may be produced as
hereinbefore described and then genetically modified.
The pluripotent cells may be of any suitable type, as hereinbefore
described.
The present invention also provides a method of producing genetically
modified EPL cells, which method includes
providing
pfuripotent cells, and
CA 02324591 2000-09-19
WO 99153021 PCTlAU99/00265
24 -
a biologically active factor or large or low molecular weight
component thereof according to the present invention, or
a conditioned medium according to the invention, or
an extracellular matrix according to the invention and optionally the
low molecular weight component of the biologically active factor according
to the present invention;
modifying one or more genes in the pluripotent cells; and
contacting the genetically modified pluripotent cells with the biologically
active factor or the large or low molecular weight component thereof, or the
conditioned medium, or the extracellular matrix; in the presence or absence of
additional gpi 30 agonist to produce the genetically modified EPL cells.
The pluripotent cells may be of any suitable type, as hereinbefore
described.
Alternatively, EPL cells may be produced as hereinbefore described and
then genetically modified.
The method may include the further step of differentiating the genetically
modified EPL cells to produce genetically modified partially or terminally
differentiated cells.
Alternatively genetically modified EPL cells andlor ES cells may be
produced by dedifferentiating genetically modified partially or terminally
differentiated cells by a method including:
providing
partially or terminally differentiated cells ; and
a biologically active factor or large or low molecular weight
component thereof according to the present invention, or
a conditioned medium according to the invention, or
an extracellular matrix according to the present invention and
optionally a low molecular weight component of a biologically active factor
according to the invention;
modifying one or more genes in the partially or terminally differentiated
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
cells; and
dedifferentiating the genetically modified partially or terminally
differentiated
cells, by methods that include contacting the genetically modified
dedifferentiated
cell with the biologically active factor or the large or low molecular weight
5 component thereof, or the conditioned medium, or the extracellular matrix;
in the
presence or absence of additional gp130 agonist to produce the genetically
modified EPL cells.
The genetically modified EPL cells so formed can be reverted to genetically
modified ES cells and/or differentiated to form genetically modified partially
or
10 terminally differentiated cells as hereinbefore described.
in one aspect of this method, genetically modified partially or terminally
differentiated cells can be used as karyoplasts in nuclear transfer to form
genetically modified dedifferentiated pluripotent cells. The dedifferentiated
pluripotent cells so formed can be used to produce genetically modified ES
cells
15 and EPL cells by methods that include contact with
a biologically active factor or large or low molecular weight component
thereof according to the present invention, or
a conditioned medium according to the present invention, or
an extracellular matrix according to the present invention and optionally a
20 low molecular weight component of a biologically active factor according to
the
invention;
in the presence or absence of additional gp130 agonist.
The genetically modified ES and EPL cells so formed can be differentiated
to form genetically modified partially or terminally differentiated cells as
25 hereinbefore described.
Modification of the genes of these cells may be conducted by any means
known to the skilled person which includes introducing extraneous DNA,
removing
DNA or causing mutations within the DNA of these cells. Modification of the
genes includes any changes to the genetic make-up of the cell thereby
resulting in
a cell genetically different to the original cell.
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/002b5
26 -
Accordingly, this method provides a pluripotent cell or genetically modified
pluripotent cell which has been maintained in its pluripotent state or a
genetically
modified partially differentiated cell which has been maintained in its
partially
differentiated state and which may be used to form chimeric animals and
transgenic animals, including animals generated by nuclear transfer.
In a further aspect of the present invention there are provided ES cells,
genetically modified ES cells, EPL cells, genetically modified EPL cells,
partially or
terminally differentiated cells and genetically modified partially or
terminally
differentiated cells produced by the methods of the present invention.
In a further aspect of the present invention, there is provided a method of
producing a chirneric animal said method including
providing
a pluripotent cell or a genetically modified pluripotent cell according
to the present invention, and
a pregastrulation embryo; .
introducing the pluripotent cell or genetically modified pluripotent cell into
the pregastrulation embryo; and
monitoring chimera forming ability. _.
Accordingly, in a still further aspect of the present invention there is
provided a chirnaeric or transgenic animal, including animals derived by
nuclear
transfer, produced using a cell or genetically modified cell according to the
present
invention.
The biologically active factor, components thereof, conditioned medium,
extracellular matrix, cells and methods of the present invention have a number
of
applications, including the following:
(1 ) Source of cytoplasts or karyoplasts in nuclear transplant. For example:
~ EPL cells of any origin, and cells derived from EPL cells (ES cells,
partially differentiated cells, terminally differentiated cells) may be
CA 02324591 2000-09-19
WO 99/53021 PC'TIAU99/00265
27 -
used as cytoplasts or karyoplasts in nuclear transfer.
~ Genetically modified ES cells, genetically modified EPL cells, and
genetically modified partially differentiated and genetically modified
terminally differentiated cells may be used as cytoplasts or
karyoplasts nuclear transfer.
~ EPL cells derived by dedifferentiation of partially differentiated cells
or terminally differentiated cells may be used as source of nuclear
material for nuclear transfer.
~ Genetically modified EPL cells derived by dedifferentiation of
genetically modified partially differentiated cells or genetically
modified terminally differentiated cells may be used as source of
nuclear material for nuclear transfer.
~ Unmodified or genetically modified pluripotent cells, partially
differentiated cells, terminally differentiated cells, tissues, organs and
animals may be derived by nuclear transfer when EPL cells are used
as cytoplasts or karyoplasts.
(2) Use in human medicine. For example:
~ EPL cells obtained from any source, and preferably their
differentiated progeny obtained by programmed or directed
dififerentiation may be used in unmodified form for human cell
therapy.
The preferred mode of use is to use autologously-derived EPL cells
and their progeny.
- Cells programmed to form ectodermal lineages can be used for
cell therapy procedures including but not restricted to neuronal
and dermal cell therapy procedures. For example cell therapy
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
28 -
using neuronal cells can be used to treat Parkinson's disease.
- Cells programmed for mesodermal lineages can be used for
cell therapy procedures including but not restricted to bone
marrow and muscle cell therapies; for example, for the
treatment of cancer and for bone marrow rescue.
~ Genetically modified EPL cells obtained by any means described in
this application and preferably their differentiated progeny obtained
by directed or programmed differentiation may be used for human
gene therapy.
Such gene therapy would preferably be conducted using
autologously-derived EPL cells or their differentiated progeny.
- Examples of genetic diseases that could be treated include but
are not restricted to haemophilia, diabetes type 1, Ducheynne's
and other muscular dystrophies, Gauche's disease and other
i5 mucopoiysaccharide diseases, cystic fibrosis.
EPL cells obtained from any source, and preferably their
differentiated progeny obtained by programmed or directed
differentiation may be genetically modified to render them resistant
to viral infection by genetic manipulation of for example viral
receptors to provide cells conferring resistance to infection by that
virus.
~ EPL cells obtained from any source, and preferably their
differentiated progeny obtained by programmed or directed
differentiation may be genetically modified to allow the cells to act as
drug delivery systems for biologically active protein drugs such as
cytokines or lymphokines for example interleukin-2 for treatment of
cancers and other diseases.
CA 02324591 2000-09-19
WO 99!53021 PCT/AU99/00265
29 -
~ Unmodified or genetically modified EPL cells obtained from any
source, and preferably their differentiated progeny obtained by
programmed or directed differentiation may be used to generate cells
and tissues and components of organs for transplant.
The present invention will now be more fully described with reference to the
accompanying exampies and drawings. It should be understood, however, that
the description following is illustrative only and should not be taken in any
way as
a restriction on the generality of the invention described above.
In the figures and tables:
Figure 1. MEDII effects the transition of ES cells to EPL cells. ES cells
grown in medium containing LIF (A), 50% MEDII + LIF (B) or no additives (C)
after
4 days in culture. ES cells cultured in the presence of MEDII show a
characteristic
morphology associated with the formation of EPL cells. The bar indicates 50
u.m.
(D) ES cells were seeded at a density of 250 cells/cm2 in DMEM containing LIF,
50% MEDII+LIF, 50% MEDII and without addition. After 5 days the cultures were
stained for alkaline phosphatase and haematoxylin, and the percentage of ES,
EPL and differentiated colonies determined. Alkaline phosphatase positive
colonies were subdivided into ES and EPL cell colonies on the basis of
morphology, differentiated colonies (D) were determined as alkaline
phosphatase
negative. Plating efficiencies (%) for each condition were 42.8 +I- 8.7 (LIF);
47.26
+I- 2.6 (50% MEDII+LIF); 40.5 +I- 5.9 (50% MEDII); 41.4 +l- 6.6 (no addition).
Figure 2. (A) Northern blot analysis of ES and EPL cell RNA. 20 pg of total
RNA, isolated from E14 ES cells, EPL cell derivatives cultured in MEDII or
MEDII+LIF for 2, 4, 8, and 16 days (i), and spontaneously differentiated ES
cells
cultured for 6 days in the absence of exogenous factors (ii), was anaiysed for
the
expression of FgfS, Rexl, Oct4, and mGAP. FgfS transcripts were 2.7 and 1.8 kb
(Hebert et al., 1990), Rex1 1.9 kb (Hosler et al., 1989), Oct4 1.55 kb (Rosner
et
al., 1990) and mGAP 1.5 kb. (B,C) In situ analysis of ES (B) and EPL (C) cell
layers for the expression of Oct4 . (D,E) ES (D) and EPL (E) cells were
stained for
the presence of alkaline phosphatase. (F) 10 pg RNA from ES celis and EPL
cells
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
30 -
cultured for 2, 4 and 6 days in MEDII+LIF and MEDII was analysed for the
expression of Gbx2, Ocf4 and mGAP by RNase protection. {G) In situ analysis of
EPL cells for the expression of FgfS. (N) 20 p.g of total RNA from ES cells
and
EPL cells maintained in MEDII+LIF for 2 and 6 days was analysed by northern
blot
for the expression of uvomorulin {Uvo). mGAP expression was used to normalise
RNA levels. The bars indicate 32 u,m in B-E and 25 p.m in F.
Figure 3. Sequence of L 17 (A), K7 (B) and Psc7 {C) ddPCR products.
Figure 4. Northern analysis of L i7 (A), Psct (B) and K7 (C) expression in 5
pg of poly A RNA {L77 and Psc1) or 20 p.g total RNA {K~ isolated from ES cells
and EPL cells, maintained in culture in MEDII + LIF for up to 8 days.
Transcript
sizes are indicated in the figure. Northern blots were quantified and
normalised to
the expression of Ocf4.
Figure 5. In vivo expression of novel marker genes, L17 (A), Psc1 (B) and
K7 (C) by wholemount in situ hybridisation analysis of mouse embryos dissected
between 3.5 d.p.c. and 5.5 d.p.c.. PE - primitive ectoderm; PC - proamnionic
cavity; VE - visceral endoderm.
Figure 6. hLIF is required for the maintenance of EPL cells in culture. ES
cells were seeded at a density of 250 cellslcm2 into DMEM containing 50% MEDII
or 50% MED11+mLIF (1000 unitslml), hLIF (1000 units/ml), anti-hLIF antibodies
(10
Nglml), hLIF (1000 units/ml) and anti-hLIF antibodies (10 pglml), mLIF (1000
units/mi) and anti-hLIF antibodies (10 Ng/ml), anti-gp130 antibodies (10
Nglml) or
hLlF (1000 units/ml) and anti-gp130 antibodies (10 Nglml). After 5 days in
culture
colonies were stained for alkaline phosphatase and the percentage of EPL cell
containing colonies (EPL) and differentiated colonies (D) determined. Plating
efficiencies (%) for each condition were 32.5 +I- 1.5 (50% MEDII); 39.8 +I-
1.3
(50% MEDII+mLIF); 35.0 +l- 0.2 (MEDII + hLIF); 25.5 +I- 1.0 (MEDII + anti-hLIF
antibodies); 28.8 +I- 3.8 (MEDII + hLiF + anti-hLIF antibodies); 40.0 +/- 0.5
(MEDII
+ mLIF + anti-hLIF antibodies); 36.8 +/- 3.3 (MEDII + anti-gp130 antibodies);
3fi.3
+I- 2.3 {MEDIC + hLIF + anti-gp130 antibodies).
CA 02324591 2000-09-19
WO 99/53021 31 PCT/AU99/00265
Figure 7. (A) 5 u.m sections stained with haemotoxylin:eosin of aggregates
formed from ES cells and grown in DMEM {EBs, i) or DMEM:MEDII (EBMs, ii) for
4 days. (B) Aggregates formed from ES cells and grown in DMEM {EBs, i, iii, v)
or
DMEM:MEDII (EBMs, ii, iv, vi) for 4 days were analysed for expression of Oct4
(i,
ii), brachyury (iii, iv) and FgfS (v, vi) by wholemount in situ hybridisation.
Figure 8. 20 Ng of total RNA isolated from EBs and EBMs on days 2, 3 and
4 of development, was analysed for the expression of FgfS, brachyury, Oct4,
and
mGAP by Northern blot analysis. FgfS transcripts were 2.7 and 1.8 kb (Hebert
et
al., 1990), brachyury 2.1 kb (Lake, 1996), Oct4 1.55 kb (Rosner et al., 1990)
and
mGAP 1.5 kb.
Figure 9. (A} Northern blot analysis of EPL cells and reverted EPL cells.
ug of total RNA from EPL calls, cultured in the presence of MEDII or
MEDII+LIF for 2, 4 and 6 days, and their reverted derivatives, reverted in
medium
containing LIF alone (2R, 4R and 6R), was analysed by Northern blot for the
15 expression of FgfS, Rext, Oct4 and mGAP. (B) Clonal EPL cell lines #1 and
#2
were seeded at a density of 250 celislcm2 and 500 cells/cm2 respectively into
medium containing mLIF and 50% MED11 + LIF. After 5 days the cultures were
stained for alkaline phosphatase and the percentage of ES, EPL arid
differentiated colonies was determined. Alkaline phosphatase positive colonies
20 were subdivided into ES and EPL cell colonies on the basis of morphology,
differentiated colonies {D) were determined as alkaline phosphatase negative.
Plating efficiencies (%} were 18.4 +I- 2.fi2 (clone #1, LiF); 34.8 +I- 4.56
{clone #1,
50% MEDII + LIF); 20.4 +I- 1.71 (clone #2, LIF); 31.16 +I- 2.93 (clone #2, 50%
MEDII + LIF).
Figure i0. (A) 20 erg of total RNA isolated from ES cells (day 0) and ES cell
EBs on days 1, 2, 3 and 4 of development was analysed for the expression of
Rexl, FgfS, Oct4 and mGAP by northern blot analysis. (B) The number of
alkaline
phosphatase positive pluripotent cell colonies derived from culture of single
cell
suspensions of day 5, 6, 7 and 8 EBs in DMEM supplemented with LIF {grey) or
DMEM supplemented with LIF + 50% MEDII (black).
SUBSTITUTE SHEET (Rule 26) (ROIAU)
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
32
Figure 11. Maintenance of embryo-derived primitive ectoderm in culture
requires MEDII. (A) Morphology of 5.5 d.p.c. embryo explants cultured for 5
days
in embryo culture medium (LIF/C1V) or embryo culture medium + 50% MEDII
(MEDIIICIV}. (B) In situ hybridisation analysis of Oct4 expression in 5.5
d.p.c.
embryo explants cultured for 5 days in embryo culture medium + 50% MEDII. The
same explant is shown in both panels, with additional 4x magnification in the
right
panel.
Figure 12. (A) Schematic representation of the strategy used for isolation
and maintenance of pluripotent cells from the primitive ectoderm of the mouse
embryo. (B) Embryonic explants representative of different stages of the
isolation
procedure. Expiants from several different isolation experiments are shown.
Figure 13. Pluripotent cell colonies isolated from the primitive ectoderm of
5.5 d.p.c. embryos (EEPL cells}, and established in long term culture on STO
feeder layers, were analysed for expression of alkaline phosphatase and Oct4
by
cell staining and in situ hybridisation.
Figure 14. Two soluble factors within MEDII are responsible for the
formation of EPL cells from ES cells. sfMEDiI was separated via
ultrafiltration on
a centricon-3 unit into a retained fraction (R) and an eluted fraction (E}. ES
cells
were seeded at a density of 250 celUcrn2 into medium containing E, R or E + R
in
the presence of LIF. After 5 .days the cultures were stained for alkaline
phosphatase and the percentage of ES, EPL (alkaline phosphatase positive) and
differentiated colonies {alkaline phosphatase negative; D) was determined.
Plating efficiencies were 48.4% (eluted fraction}; 41.6% (retained fraction);
49.2%
{eluted and retained fractions); 32.8% (mLIF).
Figure 15. Purification of the tow molecular weight component of the EPL
cell-inducing activity. (A) Fractionation of E by Sephadex G10 gel filtration.
(B)
Fractionation of active fractions from A by normal phase chromatography. (C)
Fractionation of active fractions from B by Superdex peptide gel filtration.
Eluted
material was detected by absorbance at 215 nm. Chromatographic fractions were
assayed for the presence of the low molecular weight component of the EPL cell-
CA 02324591 2000-09-19
WO 99/53021 PCTIAU99/00265
33 -
inducing activity. Solid lines beneath each chromatogram indicate fractions
containing this activity.
Figure 16. Purification of the large molecular weight component of sfMEDlI
- protocol 1. Sequential purification of R via anion exchange (A), hydrophobic
interaction (B), heparin affinity (C) and Superose 6 gel filtration (D)
chromatography. Protein was detected at 280 nm. A solid bar indicates
fractions
containing the bioactivity. (E) Silver stained reducing SDS 10% acrylamide gel
of
active fractions from each stage of purification. Samples contained either 5
p.g
(1 OOKD; Anion ex.; Hydrophobic interaction) or 2 p.g (Heparin affinity; Get
filtration)
protein.
Figure 17. Purification of the large molecular weight component of sfMEDlI
- protocol 2. Sequential purification of R by Heparin Sepharose GL-6B (A),
anion
exchange (B) and Superose 6 gel filtration (C) chromatography. Protein was
detected at 280 nm. A solid bar indicates fractions containing the
bioactiv'ity. (D}
Silver stained reducing SDS 7 0% acrytamide gel of active fractions from each
stage of purification. Samples contained either 5 p,g (sfMEDlI), 2 pg (Heparin
affinity; anion exchange) or 1 pg (gel filtration) protein.
Figure 18. Purification of the targe molecular weight component of sfMEDlI
- protocol 3. (A) Purification of sfMEDlI by gelatin Sepharose affinity
chromatography. Protein was detected at 280 nm. Elution of bioactivity is
indicated by the solid bar. (B) Silver stained reducing SDS 10% acrylamide gel
of
5 pg protein from sfMEDlI and flow fraction of gelatin Sepharose and 1 pg of
cellular fibronectin purified from stromat cells (cFN Stromat cells) and
gelatin
Sepharose purified large molecular weight component from sfMEDlI (cFN SFM2).
Figure 19. Morphology of ES cells cultured in (A) human plasma ftbronectin
(2 p.glml), (B) human cellular fibronectin (2 p.glml) and (C) the large
molecular
weight component of sfMEDlI purified by protocol 2 {2 p.glml) in the presence
of
87 IrM L-proline.
CA 02324591 2000-09-19
WO 99/53021 34 PCT/AU99/00265
Figure 20. Western blot of purified human plasma fibronectin (1 ~.g, 0.5
pg), human cellular fibronectin (1.0, 0.5, 0.25 p,g) and the large molecular
weight
component of sfMEDlI purified by protocol 2 (1.0, 0.5, 0.25 pg) probed with a
monoclonal antibody (3E2, Sigma) directed against human cellular fibronectin.
The positions of protein size markers are indicated.
Figure 21. Components of the EPL cell-inducing activity can be identified in
divergent cell types and species. (A) Morphology of ES cells seeded into DMEM
+
50% conditioned medium from primary adult mouse or embryonic chick
hepatocytes after 3 days in culture. (B) Morphology of ES cells seeded into
DMEM + 50% conditioned medium from Hepa-1 c1 c 7 cells +I- 40 NM L-proline, or
DMEM + 50% conditioned medium from END-2 cells +/- 10 pg cellular fibronectin.
(C) 5 p.g protein from DMEM conditioned by 1, no cells; 2, Hep G2; 3, Hep 3B;
4,
Hepa-1 c1 c 7; 5, END-2; 6, PYS-2 electrophoresed on a 10% reducing SDS
polyacrylamide gel. Proteins were visualised by silver staining. The arrow
indicates the position of cellular fibronectin monomers.
Figure 22. Morphology of ES and EPL cell EBs at day 4 of differentiation.
(A) ES EBs, (B) EPL EBs. (C, D) EBs were sectioned and stained with
toluidine-blue; (C) ES EB, (D) EPL EB.
Figure 23. Expression of the pluripotent cell markers FgfS, Rexl, and Oct4
in differentiating ES and EPL cell EBs. Northern blot analysis was carried out
on
20 pg total RNA from ES and EPL cell EBs at indicated days of differentiation.
Expression of mGAP was used as a loading control.
Figure 24. EPL cells form parietal endoderm but not visceral endoderm
when differentiated as EBs. (A) Expression of SPARC in EBs derived from ES
and EPL cells at days 1-4 of differentiation as determined by northern blot
analysis on 20 Ng total RNA. (B, C) Sections of day 4 ES (B) and EPL (C) cell
EBs subjected to wholemount in situ hybridisation with antisense DIG-labelled
SPARC-specific riboprobes. (D, E) Whofemount in situ hybridisation of day 4 ES
(D, F} and EPL (E) cell EBs with antisense DIG-labelled AFP-specific
riboprobes.
Areas expressing AFP are indicated with arrows. (F) Cross section of an ES
cell
SUBSZITtTTE SHEET (Rule 26) (RO/Ain
CA 02324591 2000-09-19
WO 99!53021 35 PCT/AU99100265
expression.
Figure 25. EPL cells regain the ability to form visceral endoderm when mixed
with ES cells during EB formation. Day 4 chimaeric EPL(LZ+):ES EBs formed by
1:1
cell mixing were stained for ~-galactosidase activity (cells within circled
area),
sectioned and stained for AFP protein by imrnunohistochemistry (black).
Figure 26. Enhanced and accelerated mesoderm induction in EPL cell EBs
compared to ES cell EBs. (A) Expression of brachyury, goosecoid and mGAP in
ES and EPL cell EBs at day 0-4 of differentiation as determined by Northern
blot
analysis on 20 Ng total RNA. (B-1) Wholernount in situ hybridisation of ES
(B,D,F,H) and EPL (C,E,G,I) cell EBs with antisense DIG-labelled riboprobes
specific for brachyury (B-E) and Ocf4 (F-1 ) at day 3 (B,C,F,G) and day 4
(D,E,H,I).
Figure 27. Enhanced and accelerated formation of beating cardiocytes in
EPL cell EBs compared to ES cell EBs. (A) The percentage of ES and EPL cell
EBs exhibiting beating muscle during days 4 to 12 of differentiation. The mean
percentage and s.d. were derived from two independent experiments in each
experiment n=48. (B) Nkx2.5 expression in ES and EPL cell EBs at day 4-12 as
determined by Northern blot analysis on 20 pg total RNA.
Figure 28. The ability of EPL cells to form neurons depends on the in vitro
differentiation regime employed. (A) Percentages of ES and EPL cell EBs
forming
neurons on days 8, 10 and 12 of differentiation. (B) Percentages of ES and EPL
cell RA-treated aggregates containing neurons. The mean percentage and s.d.
was derived from four independent experiments. In each experiment n=32
Figure 29. Differentiation of ES and EPL cells in response to growth factors
of the FGF and TGF~ families. (A) An EPL cell colony, differentiated in the
presence of bFGF, showing the characteristic differentiated morphology of cell
type A. (B) The proportions of ES cell (ES), EPL cell (EPL), differentiated
cell (D)
and mixed EPL and differentiated cell (SD) colonies after culture of ES and
EPL
cells in the presence or absence of bFGF (10 nglml) after 5 days. (C) An EPL
cell
colony, differentiated in the presence of activin A, showing the
characteristic
SUBSTTTUTE SHEET (Rule 26) (RO/AU)
CA 02324591 2000-09-19
WO 99/3021 PCT/AU99/00265
36 -
differentiated morphology of cell type B. (D) The proportions of ES cell (ES),
EPL
cell (EPL), differentiated cell (D) and mixed EPL and differentiated cell (SD)
colonies after culture of ES and EPL cells in the presence or absence of
activin A
(150 nglml) after 5 days.
Figure 30. Reverted EPL cells have a differentiation potential similar to ES
cells when differentiated as EBs. (A) Northern analysis on 20 Ng total RNA of
FgfS and brachyury expression in EBs derived from ES, EPL and EPLR cells at
days 1-4. of differentiation. (B) The percentage of ES, EPL and EPLR EBs
exhibiting beating muscle from day 7 to day 12 of differentiation, n=36. (C)
The
percentage of ES, EPL and EPL~ EBs forming neurons from day 7 to day 12 of
differentiation, n=36.
Figure 31. Directed formation of pluripotent cells to neurectoderm via
formation of EBMs. (A) EBMs formed from ES cells and grown for 4 days in
DMEM + 50% MEDII and a further 3 days in DMEM + 50% MEDII + 20ng/ml
FGF4. Distinctive convoluted cell layers of pseudostratified epithelial
appearance
are formed within the aggregate. (B) Aggregates grown as for A were seeded
onto gelatin treated plastic for 16 hours in DMEM + 50% MEDII + 20 ng/ml FGF4
and analysed for the expression of Ocf4 (i), brachyury (ii), Gbx2 (iii) and
Sox7 (iv)
by in situ hybridisation.
Figure 32. Expression of neurectoderm and neural stem cell markers in
aggregates derived from EBMs. EBMs (day 4) were cultured for 3 days in DMEM
+ 50% MEDII + 20 nglml FGF4 and seeded onto gelatin for analysis of gene
expression at days 8, 9 and 10. (A) In situ hybridisation of aggregates with
Gbx2,
Sox1 and Sox2 antisense probes. (B) Immunohistochemistry of aggregates on
day 9 with antibodies directed against nestin and N-Cam.
Figure 33. Expression of pluripotent cell and neurectodeml markers during
programmed formation of neurectoderm from EBMs. (A} 10 pg total RNA from ES
cell EBs (IC+B, day, 4), ES cell EBs cultured from day 4 in DMEM + 20 nglml
FGF4 (IC+BIFGF, days 5-8), EBMs (MEDII, day 4}, and EBMs cultured from day 4
in DMEM + 50% MEDII + 20 ng/ml FGF4 (MED111FGF, days 5-8) was analysed for
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99100265
37 -
expression of Oct4, Fgf5 and mGAP by northern blot. {B) RNase protection for
Gbx2 and Sox1 on 20 erg total RNA from EBMs (day 4) and EBMs cuttured in
DMEM + 50% MEDII + 20 ng/ml FGF4 (days 6, 7, 8).
Figure 34. Formation of differentiated cell types of mesoderm and
ectoderm origin by directed differentiation of pluripotent cells in vitro.
individually
seeded aggregates developed from ES cell EBs (control EB) and EBMs (EBM)
were scored on days 8, 10 and 12 for the presence of (A) beating cardiocytes
(mesoderm-derived) and {B) neurons (ectoderm-derived).
Figure 35. The large molecular weight component of MEDII promotes
formation of neurons from EPL cells. individually seeded aggregates developed
from EPL cell EBs cultured in 1, and 4, DMEM; 2 and 5, DMEM + anti-human LIF
antibody; 3 and 6, DMEM+ anti-human LIF antibody + 100Ng/ml R were assessed
on days 7, 8, 9 and 10 for the presence of beating cardiocytes (1, 2, 3)' and
neurons (4, 5, 6).
Figure 36. Formation of mesoderm by reaggregation of EPL cells formed in
suspension. 20 pg total RNA from EBMs (day4) which had been trypsinised to
single cells and reaggregated in DMEM (IC+B) +/- 10 ng/ml FGF4, or DMEM +
50% MEDII (MEDII) +/- 10 nglml FGF4 was analysed after 2 and 4 days for
expression of brachyury and Oct4 by northern blot. Representative aggregates
in
DMEM and DMEM + 50% MEDII at day 4 are shown.
Figure 37. Nascent mesoderm in EPL cell EBs can be programmed to
formation of haemopoietic cells. ES and EPL cell EBs were cultured in
methylceliuiose supplemented with 1L-3 and M-CSF to induce formation of
macrophages. The proportion of aggregates containing macrophages was
determined on days 12, 15 and 18.
Figure 38. Comparative analysis of methodologies for directed formation of
mesoderm (EPL EBs) and ectoderm (EBM) germ layers from pluripotent cells.
Seeded aggregates were scored at day 10 for formation of beating cardiocytes
and day 12 for formation of neurons.
CA 02324591 2000-09-19
WO 99/53021 PCTIAU99/00265
38 -
Table 1. Summary of the gene expression patterns of ES cells and EPL
cells compared to gene expression in the ICM and primitive ectoderm of the
embryo. RNA was isolated from ES cells and EPL cells grown in the presence of
MEDII for 2 days and analysed by Northern blot for the expression of Oct4,
uvomorulin, FgfS, Rext, AFP, H19, Evx1 and Brachyury. Gbx2 expression was
detected using RNA protection assays. Alkaline phosphatase activity was
detected using an enrymatic stain on ES and EPL cell layers. Embryonic
expression patterns were determined by Rosner et al., 1990, Sch'ler et al.,
1990,
Yeom et al., 1991 (Oct~; Hahnel et al., 1990 (alkaline phosphatase); Sefton et
al.,
1992 {uvomorulin); Haub and Goldfarb, 1991 Hebert et ai., (Fgf5); Rogers et
al.,
1991 {Rex1); Bulfone et al., 1993; Chapman et al., 1997 (Gbx2); Dziadek and
Adamson, 1978 (AFf'); Poirier et al., 1991 {H19); Bastian and Gruss, 1990,
Dush
and Martin, 1992 (Evx1); and Herrmann, 1991 (Brachyury~.
Table 2. ES cells and reverted EPL cells, but not EPL cells, contribute to
the development of chimeric mice when injected into CBA/C57 black host
blastocysts. Summary of results from the injection of E14TG2a ES cells, their
EPL cell derivatives grown in 50% MEDII for 2 and 4 days (EPL;2 and EPL;4
respectively) and reverted EPL cells, formed by the culture of EPL cells in
medium
containing mLIF but not MEDII for 6 days (EPL;2R and EPL;4R).
-- Table 3. The effect of purified ECM components on EPL cell stability in
culture. ES cells were seeded onto tissue culture plastic pretreated with the
ECM
components gelatin, laminin, plasma fibronectin, collagen IV and a mix of
laminin,
plasma fibronectin and collagen IV in DMEM + 50% MEDII. After 5 days cultures
were stained for alkaline phosphatase and the percentages of EPL cell colonies
with no associated differentiation were determined.
Table 4. Individual 5.5 d.p.c. embryos were seeded into 2 ml collagen 1V
treated tissue culture wells in either embryo culture medium or embryo culture
medium + 50% MEDII. After 5 days explants were fixed in 4% PFA and stained
for Oct4 expression by in situ hybridisation.
Table 5. Summary of the physiochemical properties of the low molecular
CA 02324591 2000-09-19
WO 99153021 PCT/AU99100265
39
weight component of the EPL cell-inducing activity.
Table 6. Amino acid analysis of samples derived by purification (Figure 15)
of sfMEDlI (active sample) and unconditioned medium (control sample). The
sample purified from sfMEDlI was analysed with and without hydrolysis.
Table 7. Summary of the EPL cell-inducing activities of amino acids,
proline analogues and peptides tested in the presence of R. + = EPL cell-
inducing
activity (minimum active concentration identified in bold); - - lack of EPL
cell
formation; * = ES cell death at high concentrations (500 NM AZET, 100 pM 3,4
dehydro-L-proline, 50 pM sarcosine and substance P).
Tabfe 8. Summary of EPL cell formation from ES cells in response to ECM
components. ECM components were presented to ES cells in solution (+l- 40 p.M
L-proline), or as matrices (+l- 40 p.M L-proline). Purified bioactivity =
large
molecular weight component of MEDII purified by protocol 2; Medll = Hep G2
conditioned medium or ECM; + = EPL cell formation; - = no EPL cell formation;
+/- = sporadic flattened pluripotent cell colonies. ECM components were used
in
solution at 10 p,glml (collagen IV; vitronectin and plasma fibronectin) 3
p.glml
(Laminin) or 2pglrnl (cellular fibronectin and purified bioactivity). Gelatin
was
used at 0.01% and MEDII at 50%.
EXAMPLE 1
f=ormation of a primitive ectoderm tike cell population. EPL cells, from ES
cells in response to biologically derived factors
Materials and Methods
Cell culture conditions
ES cells were cultured in the absence of feeders on tissue-culture grade
plastic-ware (Falcon) pre-treated with 0.2% gelatin/PBS for a minimum of 30
minutes. Cells were cultured in Dulbecco's Modified Eagles Medium (Gibco BRL),
pH 7.4, containing high glucose and supplemented with 10% foetal calf serum
CA 02324591 2000-09-19
WO 99!53021 PC'TIAU99/00265
40 -
(FCS; Commonwealth Serum Laboratories), 40 p.g/ml gentamycin, 1 mM
L-glutamine, 0.1 mM ~i-mercaptoethanol (~i-ME), herein referred to as DMEM,
supplemented with 1000 units of LIF under 10% C02 in a humidified incubator.
Routine tissue culture was performed as described by Smith {1991). E14 ES
cells
(Hooper et al., 1987) were obtained from Anna Michelska (Murdoch Institute,
Melbourne). CCE ES cells (Robertson et al., 198fi) were obtained from Richard
Harvey {Walter and Eiiza Hall Institute, Melbourne). MBLS (Pease at al., 1990)
and D3 (Doetschman et al., 1985) ES cell lines were obtained from Lindsay
Williams (Ludwig Institute, Melbourne). CGRB and E14TG2a (Hooper et al.,
1987) ES cells were provided by Austin Smith (Centre far Genome Research,
Edinburgh).
LIF was produced from COS-1 (ATCC CRL-7 fi50) cells transfected with a
mouse LiF expression plasmid, pDRlO, as described by Smith (1991 ) with the
following modifications. COS-~ cells were transfected by electroporation using
a
Bio-rad Gene Pulsar at 270 Votts and a capacitance of 250 p,FD. Transfected
cells were plated at 7x104 cells/cm2 in DMEM, pH 7.4, containing high glucose
and
supplemented with 10% FCS, 40 pg/ml gentamycin and 1 mM L-glutamine.
Medium was collected and assayed for LIF expression as described by Smith
(1991 ). Altemativety, medium was supplemented with 1000 units of recombinant
LIF (ESGRO, AMRAD).
Hep G2 cells (Knowles et al., 1980; ATCC HB-80fi5) were maintained in
culture in DMEM and passaged at confluence. To condition medium (MEDiI) Hep
G2 cells were seeded into DMEM at a density of 5 x 104 celis/cmZ. Medium was
collected after 4-5 days, sterilised by filtration through a 0.22 urn membrane
and
supplemented with 0.1 mM ~-ME before use. MEDII was stored at 4°C for 1-
2
weeks or at -20°C for up to 6 months without apparent loss of activity.
EPL cells were formed and maintained in media containing 50% MEDII
conditioned medium in DMEM with or without the addition of L1F. EPL formation
was apparent with the addition of between 10 and 80% MEDiI, with optimal
culture
conditions at 50% MEDII (data not shown).
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
41 -
EPL cells were formed from ES cells and maintained as follows :-
Adherent culture: ES cells were seeded at a density of 1.3 x 104 cells/cm2
onto tissue-culture grade plastic-ware {Falcon) pre-treated with 0.2%
gelatinIPBS
for a minimum of 30 minutes in DMEM containing 50% MEDII as described above.
EPL cells were maintained in 50% MEDII using routine tissue culture techniques
(as described by Smith, 7 991 ).
In suspension aggregates: ES cells were seeded at a density of 1 x 10$
cells/cm2 in suspension culture in bacterial petri dishes in DMEM containing
50%
MEDII as described above. The resulting EPL cell aggregates were split 1:2
after
2 days and seeded into fresh DMEM containing 50% MEDII. Control aggregates
(EBs) were formed by aggregating ES cells in identical fashion in DMEM without
MEDiI.
DNA manipulations
All DNA manipulations were carried out using standard protocols
(Sambrook et al., 1989). DNA was sequenced using a T~ Sequenase kit
{Pharmacia) as per the manufacturers instructions. Sequencing reactions were
resolved on 6% polyacrylamide/urea gels and exposed to X-ray film as described
in Sambrook et al., 1989.
Northern Blot and ribonuclease protection analysis
Cytopiasmic RNA was isolated from cultured ES and EPL cell layers using
the method of Edwards et al (1985). Total RNA was isoiated from EPL cell
aggregates that had been washed once in 1 x PBS, pelleted by centrifugation
and
stored at -20°C. Pellets were resuspended in 1 mi of extraction buffer
(50mM
NaCI; 50 mM Tris.Cl, pH7.5; 5 mM EDTA, pH 8.0; 0.5% SDS) supplemented with
200pg proteinase K (Merck). Sterile, acid washed glass beads (0.5 mm) were
added to just below the meniscus and vortexed vigorously. A further 3 ml of
extraction bufferlproteinase K was added and incubated at 37°C for one
hour
before phenol/chforoform extraction. Nucleic acids were precipitated by the
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
42 -
addition of 400 p,l 3M NaAcetate and 4 ml of iso-propanol for 30 minutes at -
80°C
and collected by centrifugation at 3500 rprn in a benchtop centrifuge. Nucleic
acids were resuspended in 400 lr.l DNase I buffer (50 rnM Tris.Cl, pH 8.0; 1
mM
EDTA, pH 8.0; 10 mM MgCl2; 0.1 mM DTT), 20 units of RNase free DNase I
(Boehringer Mannheim) and incubated for one hour at 37°C. RNA was
precipitated by the addition of 40 l.~J 3M NaAcetate and 1 ml ethanol and
collected
by centrifugation.
Northern Blot analysis was performed as described in Thomas at al., 1995.
DNA probes were prepared from DNA tragments using a Gigaprime labelling kit
(BresaGen) or Megaprime kit (Amersham). DNA fragments were isolated form the
following plasmids. An H19 cDNA fragment was excised from LC10-8 {Poirier et
al., 1991 ) as a 778 by fragment with Pvull. A 462 by Stul cDNA fragment of
Oct4
(residues 491-593) in 8luescript KS+ was obtained from Dr. H. Scholer (Scholar
et
al., 1990). A 484 by fragment was released by XhoIIHindlll digestion and used
for
probe generation. A Brachyury specific probe was excised from pSK75 (Hemfian,
1991 ) as a 1600 by EcoRl fragment. An uvomorulin probe was excised from
F20A in pUC8 (Ringwald et al., 1987) as a 620 by EcvRt fragment. A 700 by
Pst1 /BamH1 Evx1 cDNA fragment was excised from pAB11 (Dush and Martin,
1992). Fgf5 was obtained as a full length coding region clone in Bluescript
(Hebert et al., 1990), from which an 800 by EcoRIIBamH1 cDNA fragment was
isolated and used as a probe. An AFP probe was generated from a 400 by EcoRl
fragment encoding the first 350 by of the mouse AFP cDNA cloned into in
pBiuescript KS II+ (courtesy of Dr. R. Krumlauf, NIMR, London). An 848 by Rex1
fragment, cloned into pCRT""II {pRexi , obtained from Dr. N. Clarke,
Department of
Genetics, Cambridge University, U.K.) was excised by EcoRl digestion. A mGAP
probe was synthesised by labelling a whole plasmid containing 300 by of mGAP
cDNA sequence (Rathjen at al., 1990). The 737 bp, 232 by and 458 by ddPCR
fragments of L 17, K7 and Psc7 respectively were released from Bluescript I t
KS +
by EcoR1 digest and used as probes.
RNase protection assay methodologies and antisense probes for the
detection of Gbx2 and rnGAP were as described in Chaprnan et al. (1997). An
CA 02324591 2000-09-19
WO 99153021 PCTIAU99/00265
43 -
antisense riboprobe for the detection of Oct4 was transcribed using T3 RNA
polymerase from the Oct4 containing plasmid described above which had been
linearised with Nc~l.
Northern blots and RNase protections were exposed on phosporimager
screens (Molecular Dynamics) and developed on a Molecular Dynamics Phosphor
Imager machine. Levels of gene expression were quantified using ImageQuant
(Molecular Dynamics) software.
In situ hybridisation
In situ hybridisation on cell layers, cell aggregates and whole mouse
embryos was performed using the method of Rosen and Beddington (1993} with
the following modification. Cell layers and cell aggregates were pre-
hybridised,
hybridised and washed at 60°C, and embryos were pre-hybridised,
hybridised and
washed at 65°C (L17; K7) and 63.5°C (Psc1). Cell layers, cell
aggregates and
embryos were blocked with 10% heat inactivated FCS and antibodies were added
in TBST/1 % FCS. The antibodies were not pre-adsorbed in the case of in situ
hybridisation of cell layers or ceU aggregates. Antibodies were preadsorbed
with
embryo powder (Harlow and Lane, 1988) prior to incubation with mouse embryos.
Outbred Swiss embryos were taken from time-mated Swiss female mice
on the days specified. 0.5 days post coitum was designated as noon on the day
of plugging. Mated female mice were killed by cervical dislocation or C02
asphyxiation, and uteri were kept in DMEM supplemented with 10 mM HEPES, pH
7.4 at 37°C until dissection of embryos. Embryos were removed, using
standard
dissection techniques, in PBS. Reichart's membrane was removed and the
embryos transferred directly to 4% paraformaldehyde (PFA) in PBS fixative.
Anti-sense Oct4 probes were synthesised by T3 RNA polyrnerase as
run-off transcripts from bluescript containing a 462 by Stul Oct4 cDNA
fragment
(Scholer et al., 1990), finearised with Hindlll. Sense transcripts, used as
controls,
were obtained from the same plasmid linearised with Xhol and transcribed with
T7
RNA polymerase. Sense and anti-sense FgfS transcripts were generated from a
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99100265
plasmid clone (Hebert et al., 1990) containing the full-length FgfS coding
sequence which had been linearised with either EcoRl or BamH1 and transcribed
with T7 or T3 RNA polymerase respectively. Sense and antisense probes for LT7,
Psc1 and K7 were generated from plasmid clones containing 737 bp, 458 by and
232 by fragments respectively. Antisense transcripts of L17, Psc1 and K7 were
produced by Xhol digestion and transcription with T3 RNA polymerase (Ly7,
Pscf), and HIndIll digestion with transcription with T7 RNA pofymerase (K~.
Sense probes were generated by BamH1 digestion of clones and transcription
using T7 RNA polymerase for L17 and Psc1 and T3 RNA polymerase for K7.
Antisense Brachyury probes were synthesised from pSK75 (Herrrnan, 1991 )
tinearised with BamHl and transcribed with T7 poiymerase. Sense probes were
generated from the same plasmid linearised with Hindll! and transcribed with
T3
polymerase.
Alkaline Phosphatase staining
Alkaline phosphatase was visualised using the diagnostic kit 86-R (Sigma).
The kit was used according to the manufacturer's specifications with the
following
modification; cell layers were fixed in 4.5 mM citric acid, 2.25 mM sodium
citrate, 3
mM sodium chloride, 65% methanol and 4% para-formaldehyde prior to washing
and staining.
hlistofogical analysis
Embryoid bodies were fixed in 4% paraformaldehyde (PFA) in PBS at
4°C
overnight before embedding in paraffin wax and sectioning as described in
Hogan
et al., 1994. Sections were stained with haemotoxylin:eosin.
DDPCR
Identification of transcripts differentially expressed between ES and EPL
cells by differential display PCR was performed as described in Schulz (1997)
{Psc1) and Liang and Pardee (1992) (L17, K7). Primers were designed to
CA 02324591 2000-09-19
WO 99l5302I PC'TIAU99/00265
incorporate EcoRi restriction sites and cDNA products were cloned into EcoRl
digested pBfuescript II KS +.
Results
MEDII effects the transition of ES to EPL cells in adherent culture
5 When grown in medium supplemented with recombinant LIF (LIF) and in
the absence of a feeder cell layer (Figure 1A) ES cells grow as a homogeneous
population with greater than 95% of the colonies displaying a distinctive
domed
colony morphology. To assay for factors capable of inducing ES cell
differentiation, ES cells were seeded and cultured in the presence of mLIF and
10 conditioned medium derived from mammalian cell lines. After 5 days the
cells
were assessed for divergence from the ES cell colony morphology.
When cultured in the presence of 10-80% medium conditioned by Hep G2
cells (MEDII) on tissue-culture grade plastic-ware (Falcon) pre-treated with
0.2%
gelatin/PBS ES cells gave rise to a morphologically distinct population of
cells,
15 inrhich we have termed early primitive ectoderm-like, or EPL, cells (Figure
1 B).
The formation of EPL cells was specific for MEDII and was not seen in response
to any of thirteen other conditioned media assayed. In contrast to the
characteristic ES cell colony morphology, EPL cells grew as monolayer colonies
in
which individual cells, containing nuclei with one or more prominent nucleoli,
were
20 easily discernible. Morphologically, EPL cells resembled P19 EC cells
{McBumey
and Rogers, 1982; Rudnicki and McBumey, 1987), which are considered similar to
cells of the primitive ectoderm. The culture of ES cells in the presence of
50%
MEDII resulted in a relatively homogeneous cell population in which greater
than
95% of the colonies were of the EPL cell colony morphology and in which no
25 residual ES cell colonies could be detected (Figure 1 D). This occurred in
the
presence or absence of added LIF (Figure 1D). Within the EPL cell populations
sporadic colonies of differentiated cells, similar to those seen in ES cell
cultures,
were detected {Figure 1 D). The level of differentiated colonies was 4-fold
higher
in EPL cell cultures formed in the absence of added LIF (Figure 1 D),
suggesting a
30 possible role for LIF in EPL stability. The formation of a relatively
uniform cell
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
46 _
population from ES cells contrasted with the variety of differentiated cell
types
produced when ES cells differentiate spontaneously in response to LIF
withdrawal
(Figure 1 C,D}. However, within spontaneously differentiated ES cell cultures
a
small proportion of EPL-like colonies were seen (Figure 1 D), suggesting that
EPL
cells are a normal derivative of ES cells. EPL cell morphology could be
maintained with extended culture of greater than 40 passages, or 100 days
(data
not shown) and was dependent on the continued presence of MEDII in the culture
medium. The withdrawal of MEDII and LIF resulted in the generation of an array
of differentiated cell types (data not shown) similar to those arising from
spontaneous ES cell differentiation (Figure 1C).
The transition of ES cells to EPL cells, in response to MEDII, was
demonstrated for a number of independently derived ES cell fines including
MBLS
D3, CCE, E14 and CGR8 (data not shown). The appearance of EPL cells
generated from each ES cell line was comparable.
EPL cells are pluripotent
The sporadic generation of differentiated cells in culture (Figure 1 D} and
the
spontaneous differentiation of EPL cells following withdrawal of MEDII
suggested
that these cells retained a capacity for further differentiation.
The pluripotent cells of the early mouse embryo and germ line, and ES cells
in culture, are characterised by expression of the homeobox gene Oct4 and by
alkaline phosphatase activity. Oct4 expression is restricted to the
pluripotent cells
of the early mouse embryo and is down regulated on differentiation of
pluripotent
cells both in vivo and in vitro. EPL cells were formed by culturing ES cells,
in the
presence of MEDII or MEDI I + LIF, for 2, 4, 6 and 1 fi days, with passaging
every 2
days. Northern analysis of RNA from these populations showed expression of
Oct4 at levels equivalent to the levels seen in ES cells (Figure 2Ai) and
suggested
that the formation of EPL cells is not equivalent to the process of
spontaneous
differentiation (Figure 2Aii). in situ hybridisation of ES cells and EPL cell
monolayers with an Oct4 specific anti-sense RNA probe showed Oct4 expression
un'rformly distributed across the EPL cells, and not restricted to sub-
populations of
CA 02324591 2000-09-19
WO 99!53021 PCTIAU99100265
47 -
cells within the culture (Figure 2B,C). Sporadic differentiated cells within
the
population did not express Oct4 (data not shown). Analysis of alkaline
phosphatase activity also showed uniform expression by EPL cells (Figure
2D,E},
with down regulation of activity in the differentiated cells (data not shown).
The
uniform distribution of these pluripotent cell markers in EPL cell cultures
demonstrated the homogeneity of EPL cell population.
EPL cell formation is accompanied by establishment of primitive
ectoderm-Like gene expression
The expression of pluripotent cell marker genes by EPL cells suggested
that these cells could be equivalent to a piuripotent cell population of the
early
embryo, or possibly a differentiated cell Lineage in which Oct4 expression was
not
fully down-regulated. The pluripotent cell populations of the embryo can be
discriminated from differentiated cell lineages by morphological and
developmental criteria and by the temporal and spatial expression of marker
genes. The embryonic equivalent of EPL cells was investigated by anatysis of
the
expression of marker genes that identify the pluripotent cell populations of
the
embryo, and the differentiated cells of the extra-embryonic lineages and
gastrulating embryo.
Three genes, FgfS, Rexi and Gbx2, have been reported to be differentially
transcribed between cells of the ICM and primitive ectoderm (summarised in
Table
1 ). Fgf5 expression is up regulated on the formation of primitive ectoderm
from
the 1CM, whereas both Rex1 and Gbx2 expression can be detected in the ICM but
not in the primitive ectoderm by 6.5 d.p.c.. The expression of FgfS and Rex7
in
ES and EPL cells was assessed by Northern blot (Figure 2A; Table 1 ). Gbx2
expression was analysed by RNase protection assay (Figure 2F; Table 1 ). Fgf5
expression, which was barely detectable in ES cells, was elevated 50 fold in
EPL
cells grown for 2 days in MEDII. Fgf5 expression increased in EPL cells with
time
in culture such that maximal expression, representing a 340 fold induction of
FgfS,
was reached by day 8 and persisted for at least 1 fi days in culture. Rex1 was
expressed at high levels by ES cells, but this expression was down regulated
50%
within 2 days of EPL cell formation (Figure 2A; Table 1 ). Rex1 expression was
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
48 -
reduced further in EPL cells cultured for longer periods of time such that EPL
cells
grown in MEDI1 for 6 days showed a 5 fold reduction in Rex1 expression
compared to ES cells. Gbx2 expression was high in ES cells and maintained with
2 days of culture in MEDII, but was reduced with further culture in MEDII to
undetectable levels by day 6. The changes in expression of FgfS, Rex7 and Gbx2
were delayed when the transition of ES to EPL cells was carried out in
MEDII+LIF
when compared to EPL cells formed in MEDII (Figure 2A;2F), suggesting that LIF
retarded the ES to EPL cell transition. The persistence of EPL cell morphology
and high levels of FgfS and Oct4 expression (Figure 2A) demonstrated the
ability
of MEDII to maintain EPL cells as a stable cell population and was clearly
distinct
from the gene expression changes observed during spontaneous differentiation
of
ES cells (Figure 2Aii).
EPL cell monolayers, cultured for 4 days in MEDII, were probed for FgfS
expression using DIG-labelled anti-sense FgfS transcripts. Equivalent levels
of
Fgf5 specific staining were seen in all EPL cells within the culture (Figure
2G)
confirming both the uniformity of the ES to EPL cell transition and the
homogeneity of EPL cell populations.
The expression of uvomorulin, a cadherin expressed by the pluripotent cells
of the 1CM and the primitive ectoderm but down-regulated in cells of the
primordial
germ cell lineage, was assessed in ES and EPL cell cultures by northern blot
analysis. Uvomorulin expression was maintained in EPL cells at levels
equivalent
to or greater than those observed in ES cells (Figure 2H) suggesting that
these
cells did not represent an in vitro equivalent of primordial germ cells.
Consistent with the expression pattern of pluripotent cell markers EPL cells
did not express detectable levels of marker genes specific for the extra-
embryonic
iineages of the early embryo, (H79, AFF~ the primitive streak (Evx7} or
nascent
mesoderm (Brachyury) (Table 1; data not shown).
The morphological transition of ES to EPL cells was found to be
accompanied by differential regulation of marker genes that discriminate
piuripotent cell populations in vivo. Although gene expression in ES cells was
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99I00265
49 -
equivalent to their origin from the pluripotent cells of the pre-implantation
embryo,
EPL cells expressed a repertoire of marker genes which is shared by only one
cell
population of the early embryo, the primitive ectoderm (Table 1 ).
The expression of novel pluripotent cell marker genes supports the
relationship between EPL cells and early primitive ectoderm
The transition of ES and EPL cells appears to mimic the gene expression
changes associated with the ICM to primitive ectoderm transition. A screen was
carried out to identify genes that are differentially expressed between these
cell
populations. RNA isolated from ES cells and EPL cells, cultured in MEDII+LIF
for
2, 4, fi and 8 days, was analysed using differential display polymerase chain
reaction (ddPCR). cDNA fragments expressed differentially between ES and EPL
cells were purified, cloned and sequenced.
Three novel partial cDNAs, L77, Psc) and K7, were identified {Figure 3).
Northern blot expression analysis of ES and EPL cell RNA, prepared as outlined
above, indicated that L 17 was expressed highly in ES cells but down regulated
rapidly with EPL formation and maintenance in culture (Figure 4A). Psc1 was
expressed in ES cells and down regulated in EPL cells more gradually than L17
(Figure 4B). K7 was expressed at low levels in ES cells but up regulated on
extended EPL cell culture (days 4, 6, and 8; Figure 4C).
In vivo expression of these genes in pre- and peri-implantation mouse
embryos was analysed by wholemount in situ hybridisation of mouse embryos
from 3.5 d.p.c. to 5.5 d.p.c., which encompasses the ICM to primitive ectoderm
transition (Figure 5). Expression of all three novel genes was confined .to
piuripotent cells at these stages in vivo.
L17 was expressed highly in the 1CM at 3.5 d.p.c. and in the pluripotent
cells of the ICMlepiblast at 4.5 d.p.c. (Figure 5A). L17 expression was down
regulated as the ICM/epiblast began to proliferate (day 4.75), and was not
detectable following pro-amniotic cavity formation.
CA 02324591 2000-09-19
WO 99153021 PCTIAU99/00265
50 -
Psc7 was expressed in the inner cell mass and at 3.5 d.p.c. and in the
pluripotent cells of the ICM/epiblast at 4.5 d.p.c. (Figure 5B). Unlike L17,
Psc1
expression persisted in the proliferating epiblast bud (4.75 - 5.0 d.p.c.) and
was
down regulated after the initiation of pro-amniotic cavity formation after 5.0
d.p.c.
K7 expression was not detected in the pluripotent cells of the ICM/epiblast
prior to and including 4.5 d.p.c. (Figure 5C). K7 expression was detected in
the
pluripotent cells following pro-amniotic cavity formation at 5.25 d.p.c. but
was
down regulated by 5.5 d.p.c. as the pluripotent cells reorganised to form the
columnar epithelial sheet of characteristic of primitive ectoderm.
The strict restriction of L77, Psct and K7 expression to embryonic
pluripotent cells in vivo supports the identification of EPL cells as
pluripotent.
Differential expression of these genes between the ICM/epiblast and primitive
ectoderm populations of the embryo was consistent with the identification of
EPL
cells as distinct from ES cell and more closely related to cells of the early
primitive
ectoderm.
A role for gp130 signalling in EPl. cell maintenance
The increased level of differentiated cell colonies seen in EPL cell cultures
without added mLIF when compared to cultures with added mLIF (Figure 1C,D;
Figure 6), suggested a possible role for LIF, or other cytokines which signal
through gp130, in the maintenance of EPL cells. Antibodies to mouse gpi30 (RX-
19; Koshimizu et al., 1996), which neutralise the activity of mLIF, hOSM, hIL-
6 and
hIL-11, but not hLIF, on myeloid ieukernic M1 cells and ES cells (Lake, 1996),
and
to hLIF (R&D Systems), which neutralises hLIF activity, were used to assess
the
role of gp930 signalling in EPL cell maintenance.
While EPL cell formation was observed in the presence of anti-gp130 (10
p,g/mf) or anti-hLIF (10 pg/ml) antibodies, the addition of anti-hLIF
antibodies
resulted in marked destabilisation of EPL cells compared to cells cultured in
MEDII
alone (Figure 6). This suggested that the presence of hLIF in MEDII, but not
mLIF
expressed by the cells, was important for EPL cell maintenance. The addition
of
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
51 -
both anti-hLIF antibodies and 1,000 units of mLIF (ESGRO; Amrad) reduced the
levels of differentiation to those seen in EPL cell cultures formed in
MEDII+L1F,
demonstrating that gp130 signalling was able to restore EPL cell stability in
culture.
ES cells were seeded into medium containing MEDII, anti-hLIF antibodies
(10 ~,glml) and mLIF at concentrations between 10 and 1000 unitslml (ESGRO;
Amrad). EPL cell maintenance was achieved with the addition of between 50 and
100 units/ml of mLIF (data not shown), 10 to 20 fold less than the 1000
units/ml
required for the maintenance of ES cells.
Formation of EPL cells in suspension culture
Aggregates formed from ES cells in 50% MEDII for 4 days
(EBMs) were compared to control aggregates formed from ES cells in the
absence of MEDtI for 4 days (EBs) at the level of morphology and gene
expression. EBs displayed an internal disorganisation in comparison to EBMs,
i 5 indicated by the random distribution of several internal areas of cell
death
distributed randomly throughout the EBs in comparison to a uniform central
area
of cell death in EBMs (Figure 7A). In EBMs this central area of cell death was
surrounded by an apparently homogeneous cell layer of uniform thickness.
Further, the outer layer of extraembryonic endoderm formed in ES cell EBs
could
not be discerned microscopically suggesting that EBMs were comprised of a
single cell type.
Gene expression analysis was performed by in situ hybridisation and
Northern blot analysis on EBs and EBMs developed for 4 days in culture (Figure
7B). Aggregates were analysed for expression of the primitive ectoderm markers
Ocf4 and FgfS, and brachyury, a gene up regulated on the differentiation of
pfuripotent cells into nascent mesoderm. The expression of Oct4 in EBs was
patchy, with areas of cells not staining, suggesting that a proportion of the
cells in
the aggregate had undergone differentiation. By contrast, Oct4 expression was
detected uniformly throughout the cell layer comprising the EBMs, suggesting
that
these cells remained pluripotent and had not undergone differentiation. This
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
52 -
conclusion was supported by expression of the differentiation specific marker
brachyury in approximately 10% of the EBs, suggesting that a proportion of the
cells in these aggregates had undergone differentiation to nascent mesoderm.
In
contrast, no brachyury expression was detected in the EBMs, indicating that
cells
within these aggregates had not undergone any differentiation to nascent
mesoderm.
The expression of Fgf5 in aggregates was analysed to assess the formation
of EPL celfs/primitive ectoderm (Figure 7B). in EBMs FgfS was expressed
homogeneously throughout the cell layer, indicating that the pluripoterit
cells in
these aggregates represented a uniform population of pluripotent cells which
had
undergone the transition to form EPL cells/primitive ectodem~. In contrast,
Fgf5
expression in EBs was heterogeneous.
Northern blot analysis of RNA extracted from EBs and EBMs supported the
findings of the in situ analysis (Figure 8). At day 4, EBM gene expression was
characterised by high levels of Oct4 and FgfS, diagnostic for primitive
ectoderm.
Brachyury expression, diagnostic for nascent mesoderm, could not be detected.
ES cell EBs expressed a range of genes consistent with the formation of
heterogeneous cell types.
The summation of the expression data suggested that in contrast to EBs, in
which a variety of cell types ranging from early primitive ectoderm to
extraembryonic endoderm to differentiated cells, could be detected, EBMs
consisted of a uniform population of cells equivalent to EPL cells and
embryonic
primitive ectoderm. Control aggregates, developed in 100 units of LIF without
MEDII, did not develop as EBMs demonstrating that the alterations seen in
pluripotent cell differentiation in the presence of MEDII could not be
attributed to
the low level of LIF present in MEDII (data not shown).
Summary
The data in this example demonstrate that the conditioned medium, MEDII,
contains a biological activity which directs uniform differentiation of ES
cells to an
CA 02324591 2000-09-19
WO 99l3302I PC'T/AU99/00265
53 -
alternative stable piuripotent cell population termed early primitive ectoderm-
like or
EPL cells. EPL cells were identified as most closely related to cells of the
early
primitive ectoderm based on gene expression and morphology. The transition
from ES to EPL cell could be carried out in adherent culture or in suspension
aggregates.
EXAMPLE 2
ES and EPL cells represent distinct, but interchangeable, pluripotent cell
states
Materials and Methods
All cells and tissue culture techniques were as described in Example 1
unless otherwise stated. EPL cells were reverted by seeding a single cell
suspension of EPL cells into medium containing 1000 units of LIF in the
absence
of MEDII at a density of 1.3 x 104 cells/cm2.
Blastocyst injection
ES and EPL cells were introduced to into CBA/C57 F2 blastocysts using
standard blastocyst injection technology, as described in Stewart, 1993.
GPI analysis
Glucose Phosphate Isomerase (GPI) analysis of blood and tissues was as
described by Bradley, i 987. Blood samples were collected from the tail. The
tissue samples analysed were taken from the brain, eye, femur, heart,
intestines,
kidneys, liver, lung, muscle, stomach, skin, spleen, tongue, thymus and the
ovary
or testes. Tissue samples were prepared by freeze/thawing and by
homogenisation.
Results
EPL cells seeded and cultured in medium containing LIF, but not MEDII,
CA 02324591 2000-09-19
WO 99153021 PCT'IAU99100265
54. _
were observed to adopt an ES cell-like colony morphology, a process we termed
reversion. Further, the withdrawal of MEDII, in the presence of LIF, from
established EPL cell colonies resulted in the formation of a three dimensional
colony structure suggestive of the ES cell phenotype (data not shown).
Northern
blot analysis was used to examine the expression of Oct4, FgfS and Rex7 in ES,
EPL and reverted EPL cells (Figure 9A). EPL cells cultured and passaged in
MEDII and MEDII+LIF for 2, 4 and fi days were reverted by passaging the cells
into medium containing LIF alone for 6 days. Reverted EPL cells exhibited low
Fgf5 expression and high Rex1 expression, comparable to the gene expression
profile observed in ES cells and distinct from the expression of high levels
of Fgf5
and low levels of Rex1 in the parental EPL cells. These data indicated that
the
phenotypic reversion of EPL cells was accompanied by the establishment of an
ES cell gene expression profile. These data also demonstrated a requirement
for
MEDII for both the establishment and maintenance of EPL cell characteristics.
Clonal EPL cell lines were generated and reverted to ensure that reversion
was not a consequence of residual ES cells within EPL cell populations. EPL
cells, grown for 4 days in medium containing MEDII but without added LIF, were
seeded at limiting dilution in MEDII+LIF to produce clonal EPL cell colonies.
Two
clones were expanded in MEDII+LIF over 3 weeks before seeding into medium
containing mLIF atone ar MEDII+LIF. The resulting cultures were assessed for
the presence of ES cells, EPL cells and differentiated colonies (Figure 9B). A
high
proportion of cells from both fines formed alkaline phosphatase positive
colonies
with ES cell morphology in cultures seeded and maintained in LIF alone which
were not seen in the cultures maintained in MEDII+LIF, indicating efficient
reversion of the clonal lines.
Reverted EPL cells, but not EPL cells, contribute to chimeric mice following
blastocyst injection.
Pluripotent cells from the pre- and post-implantation embryo differ by the
ability of the fomler but not the latter to contribute to embryo development
following blastocyst injection. ES cells retain the ability to contribute to
all
embryonic and adult tissues. E14TG2a ES cells and their EPL cell derivatives
CA 02324591 2000-09-19
WO 99153021 PCTlAU99/OU265
55 -
were tested for their ability to contribute to chimeric mice when injected
into
CBA/C57 F2 blastocysts. The contribution of ES cell and EPL cell derivatives
to
mouse offspring was assessed by coat colour contribution and GPI analysis of
blood and tissues. E14TG2a ES cells contributed to the development of 44% of
injected blastocysts (Table 2). fn contrast, E14TG2a derived EPL cells, grown
in
MEDII without added LIF for 2 and 4 days such that EPL cell gene expression
was
established, did not contribute to chimera fom~ation as assessed by coat
colour of
the 33 and 50 live bom pups respectively (Table 2). GPI analysis of blood
samples taken from 20 of these mice also failed to detect any contribution
from
the EPL cells. Analysis of 15 tissue samples taken from two mice also failed
to
detect any EPL cell contribution. This could not be explained by adverse
effects
of EPL cells on the viability of injected blastocysts as the percentage of
live bom
pups was comparable from blastocysts injected with ES cells and EPL cells
(Table
2).
The EPL cells used in the preceding blastocyst injection experiments were
reverted by culture in media containing LIF, but not MEDII, for 6 days {2R and
4R).
These cells contributed to embryonic development in 36% (2R} and 63% (4R) of
blastocysts injected (Table 2). GPI analysis of blood and tissue samples taken
from 10 and 2, respectively, of the chimeric mice generated using reverted EPL
cells established that these cells were able to contribute to mesodem~al,
endodermal and ectodermal derived cell lineages of the adult mouse (data not
shown). These data indicated that the ES to EPL cell transition resulted in
cells of
differing developmental capabilities, reflected in the ability of ES cells,
but not EPL
cells, to contribute to development when introduced into a host blastocyst.
The
ability of reverted EPL cells to contribute to chimera development suggests
that
this loss in chimera forming ability by EPL cells can not be attributed to a
loss of
pluripotence on EPL cell formation.
Summary
The ability of EPL cells to revert to an ES cell is consistent with the
predicted and demonstrated behaviour of other pluripotent cell types, such as
the
formation of EG cells from primordial germ cells (PGCs) in culture, and
supports
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99100265
56 -
the deduction that EPL cells are pluripotent. Further, reversion to an ES cell
type
is a predicted feature of primitive ectoderm. In combination with the
demonstrated
inability of EPL cells to contribute progeny to chimaeric mice following
blastocyst
injection, this supports the identification of EPL cells as distinct from ES
cells and
most closely related to primitive ectoderm. Finally, reversion demonstrates
that
EPL cells can be used as a substrate for the generation of ES cells in vitro
by
- dedifferentiation or reversion.
EXAMPLE 3
Isolation of EPL and ES cells from embryonic and in vitro-derived primitive
ectoderm
introduction
Existing procedures and culture conditions have failed to support the
maintenance andlor proliferation of pluripotent cells from the primitive
ectoderm of
any mammalian species. Successful isolation and mairitenance of pluripotent
cells and cell tines from the primitive ectoderm would provide an alternative
methodology for isolation of genetically manipulable pluripotent cells with
potential
for commercial, medical and agricultural application. in the mouse, such tines
have only been isolated from the piuripotent cells of preimplantation embryos
(ES
cells) or primordial germ cell lineages (EG cells). Efficient isolation of
pluripotent
cell lines is currently restricted to a limited number of inbred mouse strains
such
as 129, and has not proven successful in other mammals.
MEDII supports the formation and maintenance of EPL cells which are most
closely related to the pluripotent cells of the primitive ectoderm in the
mammalian
embryo. In this example we discuss the development of methodology which
utilises the biological activity of MEDII to isolate and maintain pluripotent
cells from
primitive ectoderm.
CA 02324591 2000-09-19
WO 99/53021 PC'f/AU99/00265
57 -
Materials and Methods
Cells and cell culture
Cell culture conditions, for ES and EPL cells are as in Example 1 except
where otherwise specified. Cells isolated from primitive ectoderm were
cultured in
Dulbecco's Modified Eagles Medium (Gibco BRL), pH 7.4, containing high glucose
and supplemented with 15% foetal calf serum (FCS; Commonwealth Serum
Laboratories), 40 fCg/ml gentamycin, 1 mM L-glutamine, 0.1 mM
~i-mercaptoethanol (~-ME), and 1000 units of LIF (embryo culture medium) under
10% C02 in a humidified incubator. Inactivated feeder layers were prepared
from
either STO cells (ATCC CRL-1503) or primary mouse embryonic fibroblasts
(Abbondanzo et al., 1993). Inactivation of feeder cells was achieved by
exposing
cells to 30 gray of ionising radiation. Feeder cells were seeded at least 6
hours
before use at a density of 1 x 105 cellslcm2 in DMEM onto tissue culture
plastic
treated with human placental collagen IV {Sigma) as per manufactureris
1 ~ instructions. Feeder layers were washed once with DMEM before the addition
of
culture medium and embryonic explants. Pluripotent cells were identified using
the piuripotent specific markers alkaline phosphatase and Ocf4 and in situ
analysis techniques as described in Example 1.
Embryoid bodies (EBs) were formed as described in Example 1.
Source of embryonic cells
CBA/C57 F2 embryos were taken from time-mated CBA/C57 F1 female
mice on the days specified. 0.5 days post coitum was designated as noon on the
day of plugging. Mated female mice were killed by cervical dislocation or C02
asphyxiation and uteri were kept in DMEM supplemented with 10 mM HEPES, pH
7.4 at 37°C until dissection of embryos. Embryos were removed, using
standard
dissection techniques, into DMEM supplemented with 10 mM HEPES, pH 7.4.
Removal of extraembryonic endoderm was achieved mechanically by pipetting
embryos through a Pasteur pipette that had been pulled in a flame to a bore
diameter slightly smaller than the embryo.
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
58 -
Results
Isolation of pluripotent cells from primitive ectoderm formed in vitro
Embryoid bodies (EBs), formed by the aggregation of ES cells in
suspension culture, follow a pathway of differentiation equivalent to early
mouse
embryogenesis with the ordered appearance of extraembryonic and differentiated
cell types. The appearance of these cells can be monitored by alterations in
gene
expression. By day 4 of EB development the pluripotent cell population within
the
embryoid body is comprised largely or entirely of primitive ectodem-~, as
determined by expression of the primitive ectoderm markers FgfS and Ocf4, and
down regulation of the ES/ICM cell marker Rex7 (Figure 1 OA). Pluripotent
cells in
EBs are largely differentiated by day 8/9 of culture.
tndividual EBs, developed for 4 to 8 days in culture, were treated with 0.5
mM EGTA in 1 xPBS for 5 minutes before being trypsinised to a single cell
suspension. Each single cell suspension was divided equally between two 2 ml
tissue culture welts containing either DMEM + LlF or DMEM + 50% MEDII + L1F.
After 5 days, cultures were stained for alkaline phosphatase activity to
identify
pluripotent cell colonies. The presence of MED11 in the medium resulted in the
isolation of numerous pluripotent cell colonies (Figure 10B), demonstrating
that
this factor allowed efficient isolation and maintenance of plurtpotent EPL
cells from
pluripotent cetfs within the EBs. These EPL cells originated from primitive
ectoderm which was shown to be the only pluripotent cell population within the
EBs at these stages by the expression of FgfS and Rex) (Figure 10A}, and by
the
failure of medium supplemented with LIF alone to support pturipotent cell
proliferation and maintenance (Figure 10B). LIF has been shown to be
sufficient
for the maintenance of pluripotent ICM or ES cells. Pluripotent cells could be
isolated from EB derived primitive ectoderm in either the presence or absence
of
LIF, but was dependent on the presence of MEDII.
These data indicate that MEDII can support the maintenance and
proliferation of pluripotent cells from the primitive ectoderm of EBs. The
pluripotent celEs isolated from the primitive ectoderm were equivalent to EPL
cells
CA 02324591 2000-09-19
WO 99/53021 PCTIAU99100265
59 -
in morphology and gene expression, and could be reverted to ES cells when
cultured in the presence of LIF and the absence of MEDII {data not shown;
Example 2). Pluripotent cells isolated from the primitive ectoderm of EBs in
this
way and reverted to ES cells have been shown to contribute to chimaeric mice
when introduced into host blastocysts.
Isolation and maintenance of pluripotent cells from primitive ectoderm of
mouse embryos
The goal of this investigation was the isolation and maintenance of
piuripotent cells from the primitive ectoderm of a non-129 strain of mice.
Successful isolation and maintenance of piuripotent cells from this strain,
which
does not normally give rise to ES cells at high frequency, would provide a
generic
technology that could be extended to other mammals.
Primitive ectoderm from 5.5 d.p.c. embryos is a preferred substrate for
pluripotent cell isolation
Whole embryos dissected from time mated female mice at 5.5, 6.5 and 7.5
(d.p.c) were dissected free of Reichart's membrane and placed individually
into 2
ml tissue culture welts, pre-treated with 0.1 % gelatin for 30 minutes, in 1
ml
embryo culture medium + 50% MEDII. After 3 days in culture at 3?°C, 10%
C02,
embryonic explants were stained for alkaline phosphatase, and the presence and
abundance of alkaline phosphatase positive cells was assessed. The number of
embryo expiants containing alkaline phosphatase positive cells was greatest in
explants of day 5.5 d.p.c. embryos (varying between 25% and 40%) and
decreased significantly with increasing age of the embryo to less than 5% in
explants of day 7.5 d.p.c. embryos. The abundance of alkaline phosphatase
positive cells in explants of 5.5 d.p.c embryos was variable, but most
explants
comprised 20% or greater alkaline phosphatase positive cells. 5.5 d.p.c.
embryos
were used as the source of primitive ectoderm in subsequent experiments.
Successful isolation and maintenance of piuripotent cells from the primitive
ectoderm has been achieved from embryos collected between 5.25 and 5.5 d.p.c..
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99100265
60 _
Collection of embryos after this window of opportunity, even by 1-2 hours, can
compromise the procedure. Explants from these embryos quickly become
differentiated and no primitive ectoderm cell layers can be identified
morphologically. The isolation of cells from embryos earlier than 5.25 d.p.c.
has
not been attempted.
Purified extracellular matrix (ECM) components stabilise EPL cells in culture
The effect of alternative extracellular matrix activities on the stability of
EPUprimitive ectoderm cells in culture was tested by seeding ES cells at low
density (2.5 x 102 cells/cm2) in DMEM + LIF or DMEM + 50% MEDII onto tissue
culture plastic which had been pretreated with gelatin (Example 1 ), plasma
fibronectin, laminin, collagen IV (all obtained from Sigma and used as per the
manufacturers specifications) and a combination of plasma fibronectin, laminin
and collagen IV. The selected purified ECM components have been shown to be
present in the extracellular matrices of the early embryo. Cells were cultured
for 5
days and stained for alkaline phosphatase. Colonies were classified on the
presence or absence of differentiated (alkaline phosphatase negative) cell
types
within the colony and the presence of differentiated cells was used as a
measure
of pluripotent cell stability (Table 3). Of the conditions tested gelatin was
the least
favourable for EPL cell stability, with 78% of EPL cell colonies containing at
least
some differentiated cells. The puri#ied matrix components all resulted in more
stable EPL cell cultures when compared to gelatin, with collagen IV and the
matrix
mix containing collagen IV resulting in 59% and 62.5% undifferentiated EPL
cell
colonies respectively. ES cells cultured in the absence of MEDII were stable
on
all the tested matrices.
Tissue culture plastic pretreated with collagen IV was used for the culture of
pluripotent cells from primitive ectoderm in subsequent experiments.
The biological activity within MEDlI is required for the proliferation and
maintenance of embryonic primitive ectoderm
The role of MEDII in primitive ectoderm maintenance in culture was
CA 02324591 2000-09-19
WO 99153021 PCT/AU99100265
61 -
assessed by placing dissected 5.5 d.p.c. embryos individually into 2 ml tissue
culture wells, pretreated with collagen 1V, in embryo culture medium or embryo
culture medium + 50% MEDII and maintained at 3?°C, 10% C02. After 5
days in
culture explants were fixed with 4% PFA and the presence of pluripotent Oct4
positive cells was assessed by in situ hybridisation (Figure 11; Table 4).
Embryos
cultured in the presence of LIF became rapidly disorganised and necrotic,
clearly
lacked a primitive ectoderm cell layer, and did not contain any Oct4 positive
cells
after 5 days. By contrast, many of the embryos maintained in the presence of
50% MEDII + LIF retained their embryonic organisation including a clearly
identifiable primitive ectoderm cell layer which stained strongly and
uniformly for
the pluripotent cell marker Ocf4 (Figure 11 B). 54% of surviving expiants
cultured
in the presence of 50% MEDII + LIF contained Oct4 positive cells indicating
that
MEDII promotes the maintenance of primitive ectoderm-derived pluripotent cells
in
culture.
Comparative analysis of multiple experiments indicated that fresh MEDtI, as
opposed to MEDII that had been frozen, was more effective for maintenance in
vitro of pluripotent cells derived from embryonic primitive ectoderm.
Other parameters pertaining to successful isolation and maintenance of
embryo-derived primitive ectoderm in vitro
The visceral endoderm is recognised as a source of inductive signals
involved in the differentiation of pluripotent cells during gastrulation.
Removal of
visceral endoderm from embryonic explants was performed to eliminate this
source of differentiation inducing signals and promote _pluripotent cell
stability in
culture. Although the effect of this step has not been quantified, successful
isolation and maintenance of piuripotent cells from the embryo has not been
achieved from explants comprising primitive ectoderm and extraembryonic
endoderm.
Further, primitive ectoderm explants which failed to adhere to the tissue
culture substratum during the initial stages of in vitro culture were shown to
contain proliferating primitive ectoderm with fewer contaminating
differentiated
CA 02324591 2000-09-19
WO 99153021 PCTlAU99100265
62 -
cells. These expiants were a preferred source of pluripotent cells for further
culture. Suspension culture of primitive ectoderm explants was achieved the
addition to 2 ml tissue culture wells of 0.5 ml 0.5% agarose in DMEM medium
base (Low melting point agarose, Sigma), which was washed 3 x 30 minutes in
embryo culture medium before being preequilibrated against embryo culture
medium containing 50% MEDII.
Isolation and maintenance of pluripotent cells from the primitive ectoderm of
5.5 d.p.c. embryos (Figure 12)
Embryos were dissected from the uteri of time mated female mice between
10 and 11 a.m. on the morning of the sixth day of gestation (approximately 5.4
d.p.c.). Each embryo was dissected to remove Reichart's membrane, the
extraplacentai cone and the visceral endoderm layer, resulting in a cup shaped
structure comprising primitive ectoderm in the absence of contaminating cell
types
(Figure 12B). All tissues were removed from the embryos mechanically.
Primitive
ectoderm explants were placed individually into 2 ml wells plugged with
agarose
and cultured in 1 ml of embryo culture medium + 50% MEDII. Embryos were
cultured at 37°C in a 10% C02 humidified incubator.
On the second or third day of culture surviving explants were removed from
suspension, redissected to remove any differentiation arising in culture and
placed
back into 2 m1 wells prepared as described above.
On day 5 of culture surviving explants (approximately 25% of explants
seeded on day 0), which usually contained identifiable epithelial sheets of
Oct4
positive cells equivalent in many respects to EPL cells (Figure 12B), were
removed from suspension, redissected again and plated onto tissue culture
plastic
pretreated with collagen IV in embryo culture medium + 50% MEDII. Explants
were maintained in embryo culture medium + 50% MEDIf for a further 2 days.
Explants at this stage were usually comprised of convoluted epithelia! sheets
(Figure 12B). Explants were then cultured in embryo culture medium in the
presence of LIF and absence of MEDII. This resulted in flattening of the
sheets
and adoption of an ES cell tike appearance (Figure 12B). Analysis of these
cells
CA 02324591 2000-09-19
~rp 99/g3pZ~ PCT/AU99/OOZ65
63 -
by in situ hybridisation showed them to be pturipotent as assessed by Oct4
(Figure
12B) and alkaline phosphatase expression.
On day 9 or 10 of culture the explants were disaggregated into single cells
and small clumps of cells by trypsinisation and seeded inta 2 ml wells
pretreated
with collagen IV or preseeded with inactivated feeder cells. The cells derived
by
this method were equivalent in appearance to ES cells (Figure 12B), expressed
the pturipotent cell markers alkaline phosphatase and Oct4 {Figure 13),
retained
the ability to differentiate spontaneously and proliferated in culture for at
least four
weeks: Pturipotent cells were isolated from approximately 10% of embryos, an
efficiency comparable with the isolation of ES cells from the 1CM of 129 mouse
btastocyst stage embryos by standard techniques.
Summary
The work described in this example provides the first demonstration that
pluripotent cells from embryonic primitive ectoderm can be isolated and
maintained in culture as pturipotent cells analogous to EPL and ES cells for
extended periods. Culture and passage of these cells was dependent on the
biological activity contained within MEDIC. Pluripotent cells obtained in this
manner are likely to be amenable to genetic manipulation and represent a
resource with potential commercial, medical and agricultural application. The
demonstration that pluripotent EPL and ES cells can be isolated from the
primitive
ectoderm of mouse species that are refractory to ES and EG cell isolation by
other
methodologies potentially provides an opportunity for the isolation of
pturipotent
cell lines from other mammalian and avian species.
The demonstration that cells equivalent to EPL cells can be isolated and
maintained from embryonic primitive ectoderm cultured in the presence of the
biological activity contained within MEDII provides further support for the
proposed
similarity between EPL cells and embryonic primitive ectoderm.
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
EXAMPLE 4
Purification of components of the biologically active factor from serum free
conditioned medium
Material and Methods
Afl cells and tissue culture techniques were as described in Example 1
unless otherwise stated. Purification of EPL cell-inducing factors utilised
standard
FPLC and HPLC chromatographic techniques with activity assessed via
morphology based assay for ES cell to EPL cell conversion. Amino acids,
proline
analogues and peptides were purchased from Sigma.
Serum free MEDII (sfMEDlI) was used as a source of the biologically active
factor in all purification protocols. SfMEDII was shown to cause the ES to EPL
cell
transition in a manner analogous to MEDII (Example 1; data not shown). To
produce sfMEDlI, Hep G2 cells were seeded at a density of 5 x 104 cellslcm2
and
cultured for three days. Cells were washed twice with 1 x PBS and once with
serum free medium (DMEM containing high glucose but without phenol red,
supplemented with 1 mM L-glutamine, 0.1 mM (3-ME, 1 x ITSS supplement
(Boehringer Mannheim), 10 mM HEPES, pH 7.4 and 110 mg/L sodium pyruvate)
for 2 hours. Fresh serum free medium was added at a ratio of 0.23 rnl/cm2 and
the cells were cultured for a further 3-4 days. SfMEDtI was collected,
sterilised
and stored as for MEDII (Example 1 ). Large-scale production of sfMEDlI was
carried out in comrgated roller bottles (Falcon}.
Cell assays for EPL cell-inducing activity
Morphology based assays to test the EPL cell-inducing activity of purified
fractions were pertormed with D3 ES cells. Assays were carried out in 2 ml
wells
(Falcon) in a total volume of 1 mUwell. ES cells (2.5x10' cells/cm') were
cultured
in the presence of semi-purified fractions and scored microscopically for EPL
cell
morphology after 4 days in culture, and macroscopically after 5 days following
staining for alkaline phosphatase activity.
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
Extracellular matrix preparations were formed as follows. Hep G2 cells
were grown to confluence under conditions described in Example 1. Cells were
incubated in 0.5% Sodium Azide/0.1 mM PMSF in PBS for 6-18 hours at
37°C or
room temperature to kill and detach cells, or cultured in 0.5mM EGTA for 15
5 minutes at room temperature to detach cells. Celts and debris were removed
by
washing with PBS. Formation of matrices from semi-purified or purified
proteins
was achieved by drying 1-2 p.g of protein onto a 2 ml tissue culture well. ES
cells
were seeded onto matrices at a concentration of 2.5x102 cells/cm2 in DMEM
supplemented with 1000 units/ml LIF and 10 p.glml L-proline. Cell morphology
10 was assayed microscopically on day 4 and following staining for alkaline
phosphatase activity on day 5.
The presence of proiine in semi-purified samples was detected directly by
thin layer chromatography of samples on 0.2mm silica gel 60 F~ plates (Merck)
in 80% propano! followed by staining with ninhydin.
15 Protease digestion
250mg Cotlagen IV (Sigma) was digested with 3 FALGPA units of
collagenase type IV (Sigma) in 50 mM Tris.C) pH 7.4, 100 rnM CaCl2 buffer in a
final volume of 10 ml and incubated overnight at 37°C with continuous
mixing.
Digested collagen fragments were separated from enzyme and undigested protein
20 by uftrafiltration through an Amicon Diaflo YM3 membrane using a 400m1
ultrafiltration cell {Arnicon) at 4°C under nitrogen pressure. The
eluate obtained
from ultrafiltration was passed through a 0.22 Irm filter and 1.5 ml aliquots
were
lyophilised and resuspended in 200 NI water. 4x50 NI of the resuspended
filtrate
was applied to a Superdex peptide gel filtration column equilibrated in water
and
25 connected to a SMART micropurification system (Pharmacia). 25 Nl fractions
were collected and assayed directly for EPL cell-inducing activity in the
presence
of R.
For trypsin digestion of the large molecular weight component of the EPL
cell-inducing activity, 80 Nl of R, at 3.6 mglml, was digested for 1 hour at
25°C with
30 100 units of trypsin (Difco) in ~20 mM Tris.Ci pH 8.5, 20 rnM CaCl2. The
reaction
CA 02324591 2000-09-19
WO 99/53021 PCTIAU99/00265
66
was stopped by the addition of 100 pM PMSF. The sample was concentrated on
a Centricon-10 column (Amicon) and assayed for the large molecular weight
component of the EPL cell-inducing activity.
Protein analysis by reducing PAGE and western blot
Protein samples were obtained directly from conditioned medium, or
chromatographic fractions were desalted on a Centricon-10 column (Amicon) and
made to the original volume with 20 mM Tris.Cl pH 8.5. Samples were
electrophoresed on 10% reducing SDS polyacrylamide gels as described by
Laemmli, 1970 and proteins were visualised by silver staining using the method
of
Heukeshoven and Derrick (1985).
For western blotting, gels were washed for 30 minutes in western transfer
buffer (39 mM glycine, 48 mM ~ Tris.Cl, 20% methanol, 0.037% SDS) and
electroblotted on a Mini-Protean ll apparatus (BioRad) in this buffer to
nitrocellulose at 400 mArnps constant voltage for 3 hours. Filters were
blocked
overnight in PBT (0.1 % Triton X-100, 1 X PBS), 5% skim milk powder.
Monoclonal
antibody 3E2 (Sigma), specific for the EDA region of cellutar human
fibronectin,
was diluted 1/1000 in 1% skim milk powder, PBT and incubated at room
temperature for 2 hours, before incubation with the filter for 2 hours at room
temperature. Filters were washed 3 times in PBT at room temperature, then
incubated for 2 hours at room temperature in goat anti-mouse alkaline
phosphatase antibody (Dako) diluted 1!10000 in 1 % skim milk, PBT. A 20 minute
wash in western buffer 1 (100 mM Tris.Cl pH 7.4, 100 mM NaCI) was followed by
2 x 20 minute washes in western buffer 2 (Tris.Cl pH 9.5, 100 mM NaCI, 5 mM
MgCl2). The reaction was developed in western substrate mix (l0ml western
buffer 2, 40u1 NBT (75 mg/ml in 70% DMF), 40u1 BCIP (50 mg/ml in 100% DMF))
in the dark and stopped by the addition of 10 ml Western buffer 1, 100 rnM
EDTA.
CA 02324591 2000-09-19
WO 99!53021 PC 1'IAU99I00265
67
, Results
Soluble biological factors within MEDII are responsible for the ES to EPL cell
transition
SfMEDII was separated into two fractions by ultrafiltration through a 10x103
M~ cut-off membrane (Centricon-3 unit; Amicon). Both fractions were assayed
for
EPL forming activity (Figure 14), defined as the ability to cause the complete
conversion of D3 ES cells to alkaline phosphatase positive EPL cells in the
presence of mLIF. ES cells seeded into the retained fraction (>3x103 M~) at
concentrations equivalent to 50% MEDII did not form EPL cells, although there
was an increase in the size of the ES cell colonies. ES cells seeded into the
eluted fraction (<3x103 M~) at concentrations equivalent to 50% MEDII gave
rise to
an array of colony morphologies including ES, EPL and differentiated cells.
The
presence of ES cells and the higher proportion of differentiated cells was
uncharacteristic of the ES to EPL cell transition, demonstrating that the
eluted
material alone was unable to induce EPL cell formation. Seeding of ES cells
into
medium containing both the retained and eluted fractions at concentrations
equivalent to 50% MEDII resulted in uniform EPL cell formation, equivalent to
that
seen for sfMEDlI. These data indicated that two separable biological factors
were
required for the conversion of ES to EPL cells.
Large scale preparation of R and E fractions from sfMEDlf
The starting material for purification and analysis of bioactive factors from
MEDII was derived by ultrafiltration of sfMEDlI over an Amicon Diaflo YM3
membrane using a 400m1 ultrafiltration cell (Amicon) at 4°C under
nitrogen
pressure. The retained fraction (R), >3x103 M~, was used immediately or
aliquoted
and stored at -20°C. The eluted fraction (E), <3x103 M~, was used
immediately or
stored at 4°C.
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
ss -
Cell assay for detection of the low molecular weight component of the EPL
cell-inducing activity
The low molecular weight component of the EPL cell-inducing activity was
assayed as described above except that 1 m! culture medium was supplemented
with 20 trl (50-100 Ng protein) of R which had been passed over a PD10 column
{Pharmacia) to remove low molecular weight contaminants.
Physiochemical properties of the low molecular weight component
The physiochemical properties of the low molecular weight component of
the EPL cell-inducing activity were deduced from treatment and assay of E
(Table
5). Activity was maintained following acid treatment at pH 2.0, repeated
freeze-
thawing, boiling for 1 hour, and reduction with 50 mM DTT at room temperature.
Although the active component was soluble in water, acetonitrile, methanol and
propanol, making it amenable to HPLC purification, it did not bind to ion
exchange
or reverse phase HPLC columns using standard techniques.
Purification of the low molecular weight component of the EPL cell-inducing
activity
220 ml of E was applied to a Sephadex G10 column (1100 ml bed vol,
110x113 mm) equilibrated in water. Elution was with water at room temperature
at
a flow rate of 35 mUminute. Fractions of 45 ml were collected and a 1 ml
aliquot
of each fraction was lyophilised. Lyophilised fractions were resuspended in
100 u.l
of water and 25 p.i was assayed for EPL cell-inducing activ'~ty. Activity was
detected in fractions 6-10, i9-25.2 minutes after injection (Figure 15A).
Fractions 7 to 9 were pooled, lyophilised and resuspended in 1 ml of 30:70
methanoi:acetonitrile. Samples were centrifuged at 14,OOOrpm for 10 minutes to
remove precipitates and applied to a 10 mm Waters radial pak normal phase
silica
column (8 mm LD.) attached to a Waters 510 HPLC machine. The column was
washed with 30:70 methanol: acetonitrile at a flow rate of 0.2 mllminute for
l5min
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
69 -
before the material was eluted with a 20min linear gradient against water
using a
flow rate of 0.5 mUminute. Eluted material was detected with a Waters 490E
programmable multiwaveiength detector set at 215 nm. 1 ml fractions were
collected, lyophilised, resuspended in 50 ~l DMEM and assayed for EPL cell
inducing activity which eluted from the column at 70% water / 30% (30:70
methanol:acetonitrile) (Figure 15B).
The fractions of highest activity from normal phase chromatography,
between 32 and 35 minutes, were lyophilised, resuspended in 50 NI water and 10
NI was applied to a Superdex peptide gel filtration column (Pharmacia)
connected
to a SMART micropurification system (Pharmacia) and equilibrated in water at
room temperature. The column was eluted with water at a flow rate of 25
pllminute (Figure 15C) and 25 NI samples were collected. This was repeated 5
times to obtain adequate sample for analysis. Individual samples were assayed
directly for bioactivity which was detected in fractions eluting approximately
71.04
to 74.04 minutes after injection in a single peak or several closely eluting
peaks (ie
fractions 8, 9 and 10). The predicted molecular weight of the active fractions
was
<700D according to the~efution volume.
Characterisation of the purified low molecular weight component.
Fraction 9 from the Superdex peptide gel filtration column was lyophilised,
derivatised with FMOC and OPA and amino acid analysis was conducted with and
without hydrolysis using a Hewlett-Packard Amino-Quant ll analyser. Results
were compared with a control sample of non-conditioned medium subjected to an
identical purification (Table 6). The amino acid alanine and the imino acid
proiine
were present in abundance compared to the control in both hydrolysed and
unhydrolysed samples. This indicates that these amino acids were present
within
the purified sample as free amino acids and not a peptide.
L-proiine (3 x 10'~M) and L-alanine (3.9 x 10~M) were assayed for EPL cell-
inducing activity. L-proline effected conversion of ES cells to EPL cells in
the
presence of R in a manner indistinguishable from the low molecular weight
CA 02324591 2000-09-19
WO 99/5302I PC1"IAU99/00265
70 _
component of sfMEDlI. This included the alterations in morphology, and FgfS
and
Rext expression reported in Example 1 (data not shown). L-aianine had no
effect
on ES cells in the presence or absence of R.
Addition of 10'~M L-proline to ES cells in the absence of R resulted in a
heterogeneous population containing some cellslcolonies morphologically
similar
to EPL cells. The addition of R was necessary for complete and homogeneous
E.PL cell formation.
The minimal active molar concentration of proline was found to be
approximately 40NM. The optimal concentration in biological assays was found
to
be 100NM.
The effect of proline analogues and proline-containing peptides on ES and
EPL cells
Proline analogues reported to have proline-like bioactivity in other cell
systems and profine-containing peptides were tested over a range of
concentrations to assess their ability to form EPL cells from ES cells in the
presence or absence of R. Results are presented in Table 7.
The proline analogues D-proline, trans-4-hydroxy-L-profine, pyrrolidine, N-
acetyl-L-proline, N-t-BOC-proline and L-pipecholic acid (PCA), had no
observable
effects on ES cell growth or differentiation in the presence or absence of R.
Sarcosine, L-azetidine-2-carboxylic acid (AZET) and 3,4 dehydro-L-proline
inhibited ES cell growth at lower concentrations and caused ES cell death at
higher concentrations without inducing EPL cell fom~ation in the presence or
absence of R. These data point to a structural requirement for proline
activity in
EPL cell formation that is distinct from that described for other profine
bioactivities.
The proline containing peptides ala-pro, gfy-pro, pro-OH-pro, ala-pro-gly,
gly-pro-ala, gly-pro-arg-pro, val-ala-pro-gly, gly-pro-gfy-gly, substance P
free acid
(arg-pro-lys-pro-gln-gln-phe-phe-gly-leu-met-OH), substance P fragment 1-4
(arg-
CA 02324591 2000-09-19
WO 99153021 PCTIAU99/OOZ65
71 -
pro-lys-pro) and Bradykinin (arg-pro-pro-gly-phe-ser-pro-phe-arg; data not
shown),
in combination with R, were able to cause conversion of ES to EPL cells at
similar
molar concentrations to L-proline (Table 7). The peptides pro-ala, pro-gly and
gly-
pro-OH-pro in the presence of R were also able to cause the conversion ofi ES
to
EPL cells, but at a molar concentration approximately 6 times higher than that
of
proline (Table 7). The lack of correlation between the specific activities and
the
molar amount of proline in these peptides provides further evidence for a
structural requirement for the action of this factor, and points to the
possibility that
it interacts with a receptor.
EPL cells were also formed when ES cells were cultured in the presence of
R and partially purified collagen IV hydrolysate, resulting from collagenase
digestion of collagen IV. Free L-proline could not be detected within the
partially
purified collagen IV hydrolysate by TLC. The bioactivity of collagen IV
hydrolysate
presumably reflects the presence of repeating Gly-Pro-X motifs in collagen IV
proteins and provides a possible source of this bioactivity in vivo by
breakdown of
ECM components.
The carboxy-terminus of substance P (fragment 5-11; gln-gln-phe-phe-gly-
leu-met-OH; data not shown), the integrin binding peptide RGD, the neurokinin
A
and B receptor antagonists (neurokinin A and senktide) and the amino acids L-
alanine and L-lysine had no observable effect on ES cells in the presence or
absence of R, and used at concentrations equal to or greater than the
bioactive
peptides described above.
At concentrations above 50 ~rM, the tachykinin substance P induced
apoptosis of ES cells. Compared to substance P free acid and substance P
fragment 1-4, substance P has a markedly increased affinity for the NK-1
receptor.
These results indicated that L-proiine and proline-containing peptides have
the bioactivity associated with the low molecular weight component of sfMEDlI.
The identification of multiple factors with similar but distinct specific
activities
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/002G5
72 -
points to structural requirement for bioactivity, suggests the involvement of
receptor interaction, and allows prediction of additional peptides with the
low
molecular component of the EPL cell-inducing activity.
Purification of the large molecular weight component of the EPL cell-
inducing activity
Cell assay for detection of the large molecular weight component of the EPL
cell-inducing activity
The large molecular weight component of the EPL cell-inducing activity was
assayed as described previously except that 1 ml culture medium was
supplemented with 500 girl of E (made to 10% FCS) or 40 NM L-proline.
Unless otherwise stated, fractions were prepared for inclusion in the assay
as follows. The protein concentration of semi-purified fractions of R from
chromatographic purification steps was estimated using a BioRad Protein Assay
kit (BioRad # 500-0001 ). Aliquots of each fraction containing 100 Ng of
protein
were concentrated on centricon-10 units (Amicon), washed with 2 ml of PBS and
reconcentrated. Each fraction was assayed for the large molecular weight
component of the EPL cell-inducing activity at a range of concentrations, from
0.1
Ng to 10 Ng of protein.
Physiochemical properties of the large molecular weight component
The physiochemical properties of the low molecular weight component of
the EPL cell-inducing -activ'ity were deduced from treatment and assay of R,
prepared as above except that an Arnicon Diaflo YM10 membrane was used for
ultrafiltration. R was passed over a PD10 column (Pharmacia) to remove low
molecular weight contaminants and used in assays at approximately 50-100
Nglml. Treatment of R by incubation at >56°C for 7 hour, or digestion
with the
protease trypsin resulted in the loss of biological activity. The large
molecular
weight component of the EPL cell-inducing activity was also unstable at pH 5.5
and below,.or following treatment with reducing agents such as 100 mM DTT for
4
CA 02324591 2000-09-19
WO 99153021 PCTIAU99/00265
73 _
hours at room temperature. These data indicated that the large molecular
weight
component of the EPL cell-inducing activity was proteinaceous.
Large molecular weight component bioactivity can be detected in the
extracellular matrix (ECM) of cultured Hep G2 cells
ES cells were seeded onto Hep G2 ECM in 2 ml tissue culture wells at low
cell density, 2.5x102 cells/cm2, and cultured for 5 days in DMEM + LIF, with
or
without L-proline. Cultures were stained for alkaline phosphatase activity
after 5
days and the cultures were assessed by morphology for EPL cell formation.
Matrix from Hep G2 cells was able to act as a source of the large molecular
weight
component of the EPL cell-inducing activity and effectively induced the
formation
of EPL cells when used in combination with L-proline (Table 8). When used
atone
the matrix was unable to induce complete conversion of ES to EPL cells,
however
a number of the colonies adopted a flattened or spread morphology.
Purification of the large molecular weight component from sfMEDlI: Protocol
1
R, prepared from 4 I of sfMEDlI as described above, was made to 20 mM
Tris.Cl pH 8.5 and passed over a 300 ml Sepharose Q anion exchange column
{Pharmacia). Bound proteins were eluted via a step wise gradient of 200 ml 20
mM Tris.Cl pH 8.5, 200 mM NaCI, 200 ml 20 mM Tris.Cl pH 8.5, 400 mM NaCI
and 200 ml 20 mM Tris.Cl pH8.5, 1 M NaCI (Figure 16A). 200 ml samples were
collected and assayed for their ability to form EPL cells in the presence of
40 NM
L-proline. Activity eluted from the column in 400 mM NaCI.
The active fraction from anion exchange chromatography was made to a
final concentration of 0.5M ammonium sulphate in 20 rnM Tris.Cl pH 8.5. The
sample was filtered through a 0.22 Nm fitter to remove precipitated.material
before
being passed over a 40 ml phenyl Sepharose fast flow hydrophobic interaction
column {Pharmacia). Bound protein was eluted from the column in a single step
with 50 ml 20 mM Tris.Cl pH8.5 (Figure 16B) and shown by assay to contain the
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
74 -
large molecular weight component of the EPL cell-inducing activity.
The active fraction from hydrophobic interaction chromatography was
diluted in 20 mm Tris.Cl pH8.5 to a conductivity below 100 mM NaCI. The sample
was passed over a 10 ml Heparin Sepharose CL-6B column (Pharmacia). Bound
protein was eluted from the column in a single step with 10 ml 0.5M NaCI, 20
mM
Tris.Cl pH 8.5 (Figure 16C) and shown by assay to contain the large molecular
weight component of the EPL cell-inducing activity.
The active fraction from Heparin Sepharose affinity chromatography was
passed down a Superose 6 gel filtration column (Pham~acia) on the SMART
system (Pharmacia) in 20 mM Tris.Cl pH 8.5, '! 50 mM NaCI (Figure 16D). 50 NI
fractions were collected and assayed directly. Fractions containing
bioactivity
equivalent to the targe molecular weight component of sfMEDlf eluted as a
single
peak between 500-1000 kDa. EPL cell fom~ation was only observed in the
presence of E or L-proline.
Reducing SDS PAGE revealed the presence of a highly purified protein of
approximately 210-260 kDa in samples containing the large molecular weight
component of the bioactivity (Figure 16E).
Purification of the Large molecular component from sfMEDlt: Protocol 2
1 Titre of sfMEDlI was applied to a 50 ml Heparin Sepharose CL-6B column
(Pharmacia) equilibrated in 20 mM Tris.Cl, pH 8.5. The column was washed
sequentially with 200m1 20 mM Tris.Cl, pH 8.5, 200 ml 20 mM Tris.Cl, pH 8.5 +
150 rnM NaCI, 200 ml 20 mM Tris.Cl, pH 8.5 + 500 mM NaCI and 200 ml 20 mM
Tris.Cl, pH 8.5 + 1 M NaCI (Figure 17A). Aliquots of each wash were assayed
for
biological activity on ES cells. The large molecular weight component of the
EPL
cell-inducing activity eluted from the column in the 500 mM NaCI fraction.
The active fraction from Heparin Sepharose affinity chromatography was
diluted to 150 mM NaCI with 20 mM Tris.Cl, pH 8.5 and applied to a 1 ml
Resource Q anion exchange column (Pharmacia) pre-equilibrated with 20 mM
CA 02324591 2000-09-19
WO 99153021 PCT/AU99I00265
75 -
Tris.Cl, pH 8.5 + 150 mM NaCI. Proteins were eluted from the column with a
gradient from 150 mM to 500 mM NaCI over 60 minutes, followed by a 5 minute
wash in 1 M NaCi at 1 ml/min (Figure 17B). Alternate 1.5 ml fractions were
assayed for the high molecular weight component of the EPL cell-inducing
activity
which eluted from this column in 200-300 mM NaCI.
Fractions 10-17 from anion exchange chromatography were pooled and
passed down a Superose 6 gel filtration column (Pharmacia) in 20 mM Tris.Cl,
pH
8.5 + 150 mM NaCI (Figure 17C). 50N1 fractions were collected and assayed
directly for the presence of the large molecular weight component of the EPL
cell-
inducing activity which eluted as a single peak between 500-1000 kDa.
The active fraction was analysed by reducing SDS PAGE and shown to
contain a highly purified protein of approximately 210-260 kDa (Figure 17D),
equivalent to the protein purified in protocol 1.
Purification of the large molecular component from sfMEDlI: Protocol 3
4 litres of sfMEDlI was applied to a 25 ml gelatin Sepharose affinity
chromatography column (Pharmacia), pre-equilibrated in PBS, at a flow rate of
approximately 5 mUminute. The column was re-equilibrated in PBS and proteins
were eluted in a 50 ml volume of 6 M urea in PBS (Figure 18A). The eluate was
dialysed against 4 x 4 litre of PBS at 4°C to remove urea. The protein
concentration of the eluate was determined using the BioRad protein assay kit
and
0.1-10 Ng was assayed for bioactivity.
The eluate was analysed by reducing SDS PAGE and shown to contain a
highly pur'rfied protein of approximately 210-260 kDa (Figure 18B), equivalent
to
the protein purified in protocols 1 and 2. The yield of purified protein was
detem~ined by Bradford analysis to be approximately 1 rngliitre sfMEDlI.
Characterisation and identification of the large molecular weight activity
The highly purified active fractions obtained from purification protocols 1-3
CA 02324591 2000-09-19
WO 99153021 PCTIAU99/00265
76 -
were able to induce EPL cell formation in the presence of E or L-proline when
used either in solution at 2 Ng/ml (Figure 19C) or when pre-coated onto tissue
culture plastic (data not shown). This is consistent with previous
identification of
the protein as a component of the ECM. EPL cell-inducing activity could not be
detected in the absence of E or L-proline.
The extra-cellular matrix proteins human laminin (Sigma), bovine vitronectin
(Dr. Z. Upton, CRC for Tissue Growth and Repair, Adelaide) collagen IV
(Sigma),
human plasma fibronectin (Sigma) and human cellular fibronectin (J-P Levesque,
IMVS, Adelaide) were tested for the ability to promote the formation of EPL
cells
from ES cells in tissue culture media in the presence of 40 ~,M proline and
compared to the active fractions (Table 8). Of these, only cellular
fibronectin at a
concentration of >_ 1 p.glml, was able to effect the formation of EPL cells
from ES
cells when added in solution (Figure 19 A, B). The activity of cellular
fibronectin,
which is a homodimer of 250 kDa disulphide bonded proteins, was consistent
with
the size and characteristics of the highly purified bioactive factor from
sfMEDlI
identified by reducing SDS PAGE.
Western blot analysis of the active fractions from Superose 6 gel filtration
(Protocol 2), using an antibody specific for cellular fibronectin, confirmed
the
identity of the 240 kDa purified protein as cellular fibronectin (Figure 20).
Multiple ECM proteins can be used to provide the large molecular weight
component of the EPL cell-inducing activity when presented to cells as a
matrix
The above mentioned basement membrane components were tested for
the ability to promote EPL cell formation when pre-coated onto tissue culture
plastic. Morphological induction of EPL cell morphology, in the presence of L-
proline, was restricted to cellular and plasma fibronectin and the basement
membrane component laminin (Table 8).
Fibronectin and laminin bind to cells via cell surface integrin receptors,
with
the specificity of ligand binding determined by various heterodimeric
combinations
CA 02324591 2000-09-19
WO 99/53021 PCTIAU99I00265
between alpha and beta integrins. These data suggest that a range of ECM
components, activating the correct integrin receptor, could induce EPL cell
formation when presented to ES cells as an extracellular matrix. However, only
cellular fibronectin has been shown to induce formation of EPL cells from ES
cells
when presented in soluble form.
Summary
Size fractionation of sfMEDlI revealed a requirement for at least two
biologically active factors in EPL cell formation, a large molecular weight
and a low
molecular weight component. The low molecular weight component of sfMEDlI
was identified as L-proline, active at concentrations of 40 ~M and above.
Analysis
of proline analogues and small peptides demonstrated that a number of small,
proline containing peptides could substitute for L-proline in the formation of
EPL
cells.
Chromatographic purification by a variety of protocols identified the large
molecular weight component of sfMEDl1 as cellular fibronectin. In the presence
of
L-proiine the large molecular weight component or cellular fibronectin was
able to
effect the fom~ation of EPL cells both in solution and as a matrix. The
ability of
several extracellular matrix components to induce formation of EPL cells when
presented to ES cells in matrix-associated but not soluble form and in
combination
with L-proline suggested that the biological activity of cellular fibronectin
was
mediated by integrins at the cell surface, most probably an alpha 7:beta 5
heterodimer.
Taken together the data presented in this example identify factors that can
induce the formation of EPL cells from ES cells when presented in soluble or
matrix associated form, and provide sufficient information for the prediction
of
factors with similar bioactivities, acting via similar receptorlmolecular
interactions.
EXAMPLE 5
Biologically active components of the EPL cell-inducing activity can be
CA 02324591 2000-09-19
WO 99/53021 PCTlAU99100265
78 -
isolated from divergent sources and species
Materials and Methods
Preparation of primary cell lines
Primary liver cells were prepared using procedures based on procedures
described by Giger and Myer (1981), with minor variations. Mice were
sacrificed
and immediately dissected to expose the liver. Each liver was perfused through
the portal vein with 10 ml PBS, followed by 10 ml 0.05% collagenase in Hanks
buffered salt solution (HBSS; 0.8g KCI, 0.12g KH2P04, 16g NaCI, 0.1 g Na2HP04,
2g glucose, 4 ml 1 % phenol red, .035g NaHC03 in 2 litres). The perfused fiver
was removed, macerated in 10 ml/iiver of 0.05% collagenase/HBSS and
incubated at 37°C for 30 minutes. . Liver suspensions were regularly
agitated by
pipetting to aid fiver disaggregation. Cells were washed twice in HBSS (10 ml)
to
remove collagenase, and contaminating eyrthrocytes were lysed in Sassa
solution
(6.95g NH4Cl, 2.0588 Tris-base, 1 g KHCO~/ litre). Following two washes in
Williamis E medium {1 mUfiver, Gibco BRt_) the cells were seeded into gelatin
treated tissue culture flasks in DMEM and incubated at 37°C, 5% C02.
Primary avian hepatocytes were prepared from 17-18 day chick embryos.
In brief, chick embryos were removed from the egg, decapitated and dissected
to
expose the heart and liver. The liver was perfused, via cannulation of the
heart,
with 10 ml of 0.9% NaCi containing 2 mM EDTA to remove blood cells, followed
by 4 m! of 0.05% collagenase in HBSS. Livers were removed, pooled in HESS
and transferred into fresh 0.05% coilagenaseIHBBS when all livers had been
perfused and collected. Pooled livers were macerated with fine tipped scissors
and incubated for 30 minutes with gentle shaking every 5 minutes and gentle
pipetting using a 10 ml pipette every 10 minutes to aid cell disaggregation.
Coliagenase was removed by washing with HBSS (2 mUliver). Contaminating
erythrocytes were iysed in Sassa solution. Following two washes with Wilfiarns
E
(2 mUiiver) to remove cell debris and haemoglobin from lysed erythrocytes, the
hepatocytes were resuspended in Williams E medium (1 mUliver) and the yield
CA 02324591 2000-09-19
WO 99/53021 PCF/AU99/00265
79 -
determined. Typically 2 x 10' cells/liver were obtained and plated out in a
175cm2
tissue culture flask and cultured in 20 ml DMEM at 37°CI5% C02.
Preparation of conditioned media
Primary mouse and chicken hepatocytes were cultured for 4 days in DMEM
before collection of conditioned medium.
The mouse hepatocellular carcinoma cell lines Hepa-1 c1 c 7 (ATCC CRL-
2026) and Hep 3B (ATCC HB-8064), and the P19 embryonal carcinoma derived
cell lines END2 (visceral endoderm like, Mummery et al., 1985) and PYS2
(parietal endoderm like, Lehman et al., 1974), were maintained in DMEM.
Conditioned medium was collected after cell lines had been cultured for 4 days
in
non-gelatinised tissue culture flasks at 37°C, 5% C02.
Conditioned medium was processed as described for MEDII in Example 1.
Conditioned medium was assayed for biological activity as described in
Examples
1 and 4.
Visualisation of bioactive components
The presence of proline in the active fraction of END-2 medium was
detected by thin layer chromatography as described in Example 4. Cellular
fibronectin was detected by reducing SDS PAGE as described in Example 4.
Results
Conditioned medium from primary mammalian and avian hepatocytes, Iiver-
derived cell lines (Hepa-1 c1 c 7 and Hep 3B) and embryonic endoderm like cell
lines (END-2 and PYS-2), were assayed for the ability to form EPL cells from
ES
cells.
Conditioned medium from the primary hepatocytes, both mouse and
chicken; was able to effect the transition of ES to EPL cells in ~cuiture
{Figure
CA 02324591 2000-09-19
WO 99!53021 PGT/AU99/00265
80 -
21A). This was only true of medium taken from seeded primary liver cells.
Passaging resulted in loss of hepatocytes, which were overgrown by cells of
fibroblastic appearance, and loss of EPL cell-inducing biological activity. No
difference in biological activity was detected in conditioned medium from
primary
embryonic (chick) and adult (mouse) hepatocytes and MEDII.
Medium conditioned by the cultured cell lines Hepa-1 ci c 7, Hep 3B, END-2
and PYS-2 did not induce the formation of EPL cells from ES cells in culture.
However, several of these cell lines could be shown to express one component
of
the biological activity. Conditioned medium from Hepa-1 ci c 7 cells, when
used in
combination with L-proline, effected the transition of ES to EPL cells (Figure
21 B),
suggesting that these cells express the large molecular weight component of
the
EPL cell-inducing activity. This was confirmed by reducing SDS PAGE analysis
of
Hepa-1 ci c 7 conditioned medium which showed the presence of a protein with
equivalent mobility to cellular fibronectin (Figure 21 C). Conditioned medium
from
Hep 3B cells was unable to effect the ES to EPL cell transition, alone or in
combination with components of the biological activity purified from MEDIC.
These
cells, and several other liver-derived cell fines did not express detectable
levels of
cellular fibronectin (Figure 21 C), indicating that expression of the large
molecular
weight component of the EPL cell-inducing activity is not ubiquitous among
liver
derived cell lines.
Conditioned medium from END-2 cells induced formation of EPL cells from
ES cells when used in conjunction with cellular fibronectin (Figure 21B),
suggesting that these cells secrete L-proline or a functional analogue but not
cellular fibronectin (Figure 21 C). Semipurification of the conditioned medium
by
normal phase chromatography (Example 4) and analysis by thin layer
chromatography demonstrated the presence of L-profine in END-2 conditioned
medium (data not shown).
Summary
Biological activities equivalent to that found in MEDII, and capable of
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
81 -
inducing the transformation of ES cells to EPL cells, have been identified in
conditioned media from cells of divergent species, including mammals and
birds,
and in tissues from both the embryo and adult. This demonstrates that the
activity has been conserved across these species during evolution and
indicates
the fundamental importance of this activity in embryonic development. Further,
individual components of the EPL cell-inducing activity are expressed by cells
derived from diverse lineages.
EXAMPLE fi
Alternative differentiation of ES and EPL cells in vitro
Materials and Methods
Cell culture and in vitro differentiation assays
All cells and tissue culture techniques were as described in Example 1
unless otherwise stated. All EPL cells used for differentiation assays were
formed
from ES cells cultured in the presence of MEDII for 2 days. The presence or
absence of additional LIF is specified for each example.
The KSF4 ES cell line, which constitutiveiy expresses nuclear localised
LacZ (Berger et al., 1995; obtained from Patrick Tam, CMIR, New South Wales,
Australia), was maintained on inactivated primary mouse embryonic fibroblast
feeder layers (Abbondanzo et al., 1993} in DMEM (high glucose) supplemented
with 40 pglml gentamycin, 20% foetal calf serum (FCS), 0.1 mM
~-mercaptoethanol, I mM L-glutamine and 1000 units LIF as before. Prior to the
formation of EPL cells, KSF4 cells were cultured on gelatin treated tissue
culture
plastic, in the absence of a feeder layer, for two passages.
For the majority of experiments detailed here, embryoid bodies (EBs) were
formed in medium without the addition of MEDII or LIF (DMEM) using the partial
trypsinisation method (Robertson, 1987). Alternatively, EBs were formed from a
single cell suspension plated at 1 x10$ cells/ml in bacteriological dishes in
ES
CA 02324591 2000-09-19
WO 99153021 PCTlAU99/00265
82 -
DMEM, or suspended in a hanging drop of medium (50 wl) at 1.fix104 on the
inside
surface of a petri dish lid. Cell aggregates formed in suspended drops were
transferred to bacteriological dishes for further culture after 48 hours. EBs
were
maintained with regular replenishing of medium. Mixed cell EBs were formed
from
a single suspension as previously described, in which the two cell populations
were mixed at the specified ratio.
Results consistent with those presented herein were obtained using EPL
cells derived from a variety of ES cell lines including, D3 (Doetschman et
al.,
1985), MBLS (Pease et al., 1990), KSF-4 (Berger et al., 1995) and E14 (Hooper
et
al., 1987), and using alternative methods of EB formation, including single
cell
suspension, hanging drop and partial trypsinisation. Furthermore the
differentiation potential reported for EPL cells was specifically associated
with
these cells as it could not be recapitulated by ES cells which had been
spontaneously differentiated by culture in the absence of LIF for equivalent
time
periods (data not shown).
Retinoic acid (RA) differentiation of pluripotent cell aggregates was carried
out by plating ES or EPL cells in bacteriological dishes, at a density of 1
x10$
cellslml, in DMEM supplemented with 1 p.M RA. After 48 hours, aggregates were
transferred to DMEM and maintained for 2 days, then seeded individually into 2
ml
tissue culture wells. The presence of neurons was assessed by nnicroscopic
examination two days after seeding. Neuron identity was confirmed by positive
staining with the neurofilament 200 antibody N-4142 (Sigma).
Cytokine/Growth Factor Assays
Assays for the effect of cytokineslgrowth factors were carried out on ES
cells and EPL cells seeded at low density (?5 celllcm2) in 4 well multidishes
(Nunc) in 0.8 ml of DMEM + LIF or DMEM + 50% MEDII + LIF respectively.
Cytokines/growth factors were added at specified concentrations and the assays
were stained for alkaline phosphatase activity after 5 days of culture as
described
in Exarnpie 1.
CA 02324591 2000-09-19
WO 99153021 PCTJAU99/00265
83 -
Gene expression analysis
RNA was isolated from ES and EPL cells using the method of Edwards et
al (1985). RNA was isotated from EBs and RA-treated aggregates by the method
of Chomczynski and Sacchi (1987).
Northern blot analysis and whoiemount in situ hybridisation were carried out
as described in Example 1. Riboprobes were synthesised as described by Kreig
and Melton (1987).
Probe fragments used for northern blot analysis and/or in situ hybridisations
were as detailed in Example 1 with the addition of; AFP, linearisation of a
ptasmid
containing a 400 by EcoRl fragment encoding the first 350 by of mouse AFP
cDNA in pBluescript KS ll+ (obtained from Dr. R. Krumiauf, NIMR, London) with
Hindlll followed by transcription with T3 polymerase; SPARC, 570 by EcoRl
fragment derived from 13643 (Mason et al., 1986); Goosecoid, linearisation of
a
plasmid containing 909 by goosecoid genomic DNA (Blum et al. 1992) with
Hindlll
and transcription with T3 polymerase; NIa2.5, linearisation of plasmid
containing
1.6 kb NIa2.5 cDNA (tints at al., 1993) with Hindlll and transcription with T3
polymerase.
Histological Analysis
EBs were fixed in 4% paraformaldehyde (PFA) in PBS at 4°C
overnight and
processed and sectioned as described in Example 1. Sections were stained with
toluidine blue for 10 seconds, cleared in Histoclear (National Diagnostics)
and
coversfipped using a xylene-based mounting medium. EBs subjected to in situ
hybridisation were fixed in 4% PFA overnight, washed several times with PBS,
0.1 % Tween-20, treated with 100% methanol for 5 minutes and then isopropanol
for 10 minutes. Bodies were then embedded and sectioned as described in
Example 1.
To detect ~-gaiactosidase activity EBs were fixed in 0.2% gluteraldehyde in
PBS for 15 minutes on ice, washed 3x15 minutes with detergent rinse (0.1 M
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
84 -
phosphate buffer (pH 7.3), 2 mM MgCl2, 0.01 % sodium deoxycholate, 0.02%
Nonidet P-450) and stained in detergent rinse containing 5 mM potassium
ferricyanide, 5 mM potassium ferrocyanide and 1 mglml X-gal for 2 hours at
37°C.
EBs were then washed 3x with detergent rinse and transferred to 70% EtOH. For
sectioning, EBs were dehydrated to 100% ethanol and processed as described in
Example 1.
Immunological detection
AFP expression in sectioned EBs was carried out essentially as described
by Dziadek and Adamson (1978} with the following modifications. Prior to
antibody incubations, endogenous peroxidase activity was blocked by incubation
of the section in 3% H202 in methanol for 30 minutes. Sections were incubated
with a 1!200 dilution of rabbit anti-AFP (ICN) for 2 hrs, washed several times
with
PBS then incubated with 1/200 dilution of HRP-conjugated anti-rabbit IgG
(Silenus).
Formation of beating cardiocytes from ES and EPL cells
Beating cardiocyte formation was assessed in individual EBs, formed from
either ES or EPL cells, plated onto pre-equilibrated agarose plugs (1 %
agarose in
DMEM medium base equilibrated against DMEM for 3 hours at 37°C, 10%
C02 in
2 ml tissue culture wells) in DMEM. The presence or absence of beating muscle
was assessed at days 4, 6, 8, 10 and 12 by microscopic examination.
Alternatively, beating muscle formation was assessed in individual EBs seeded
into 2 ml wells at day 6 of development and scored microscopically at days 7,
8,
10, 12, 14 and 16. Seeded aggregates were also scored for the presence of
terminally differentiated neurons on days 8, 10 and 12.
Results
Embryoid bodies, formed by aggregation of pfuripotent cells in suspension,
follow a pathway of differentiation reminiscent of early embryogenesis
(Example 1}
and provide an opportunity for broad assessment of pluripotent cell
differentiation
into both embryonic and extraembryonic cell types. The term EB is used herein
to
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
85 -
describe pluripotent cell aggregates cultured in DMEM.
Preliminary characterisation of EPL and ES embryoid body formation
Clear morphological differences were apparent when EBs were formed
from either ES cells or EPL cells. ES cell EBs comprised a homogeneous
population of round, relatively smooth and ordered aggregates (Figure 22A).
EBs
formed from EPL cells also formed a homogeneous population, however the
bodies appeared irregular and disorganised (Figure 22B). Sectioning and
staining
indicated that while ES cell EBs comprised a relatively uniform population of
cells
characterised by a compacted round morphology (Figure 22C}, reminiscent of
undifferentiated piuripotent cells, internal Celts in EPL EBs were loosely
packed
and heterogeneous, possibly reflecting differentiation (Figure 22D}. This
observation prompted a detailed comparative investigation of ES and EPL cell
. differentiation.
Accelerated pluripotent cell differentiation in EPL cell embryoid bodies
The progression from ICM to primitive ectoderm to germ layer formation
can be monitored via alterations in gene expression both in vivo and during
embryoid body differentiation in vitro. Rexl is expressed by ICM and ES cells,
and is down regulated in EPL cells, the primitive ectoderm at 6.0 d.p.c. and
during
ES cell differentiation in vitro. Fgf5 is not expressed by ICM or ES cells,
but is
expressed in EPL cells and the primitive ectoderm prior to and during
gastrulation,
and up regulated transiently during ES cell differentiation in vitro. Ocf4 is
expressed by all pluripotent cell populations of the embryo, EPL cells and ES
cells, and is down regulated upon differentiation of these cells in vivo and
in.
Although the expression of Rex7 was found to be down regulated in both
ES and EPL cell embryoid bodies, the kinetics of Rexl regulation differed
between the two cell populations. Down regulation to a barely detectable,
basal
level was observed by day 1 in EPL cell embryoid bodies (Figure 23). In
contrast,
a similar level of Rex1 expression was not observed in ES cell embryoid bodies
until day 2, with intem~ediate levels of expression observed on day 7 .
Similarly,
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
86
the kinetics of FgfS regulation differed between embryoid bodies derived from
ES
or EPL cells. FgfS, already expressed in EPL cells, was up regulated rapidly
from
day 1 in EPL cell embryoid bodies to a peak on day 2/3, followed by a marked
decrease in expression on day 4. Up regulation of FgfS expression in ES cell
embryoid bodies was not observed until day 3. While expression levels in these
bodies increased further at day 4, they remained below Fgf5 levels in EPL cell
bodies at days 2 and 3. Ocf4 expression decreased with time in both ES and EPL
cell embryoid bodies but was down regulated more rapidly in EPL cell embryoid
bodies, with a 4 fold decrease seen between day 3 and 4 (Figure 23). This
decrease in Oct4 expression followed the highest levels of FgfS expression and
presumably reflected differentiation of pluripotent cells within the bodies.
The changes in Rexl, FgfS and Oct4 expression observed in both ES and
EPL embryoid body development suggested that differentiation reflected the
events of normal embryogenesis and proceeded through the formation of fate
stage primitive ectoderm. Further, the rapid increase in FgfS expression and
the
earlier onset of differentiation, as detected by the loss of Oct4 and FgfS
expression, indicated that pluripotent cell differentiation within EPL cell
ernbryoid
bodies was accelerated cornpared to ES cell embryoid bodies. These properties
are consistent with our previous alignment of ES and EPL cells with
pluripotent
cell populations occurring prior to 6.0 d.p.c.
The EPL cell embryoid body environment is non-permissive for visceral
endoderm formation
By 4.5 d.p.c. pluripotent cells exposed to the blastocoelic cavity have
differentiated to form primitive endodemt. The primitive endoderm gives rise
to
two distinct endodermal cell populations, visceral endodem~, which remains in
contact with the epiblast, and parietal endoderm, which migrates away from the
pluripatent cells to form a layer of endoderm adjacent to the trophectoderm.
While others have analysed extraembryonic endodem~ fom~ation at relatively
late
stages (day 9-12) of embryoid body development, formation of an outer layer of
endoderm, containing both visceral and parietal endodeml cells, can be
observed
in embryoid bodies by day 4 or 5 of development. This timing is coincident
with
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/OOZ65
87 -
fomlation of primitive ectoderm and creation of an inner cavity, events that
accompany endoderm specification in the embryo. Analysis of endoderm
formation at this stage is therefore more likely to reflect nom~al embryonic
events.
Fom~ation of parietal and visceral endoderm during EPL and ES embryoid
body development was analysed by SPARC and AFP expression respectively.
SPARC expression followed similar kinetics in ES and EPL cell embryoid bodies,
with an approximately 2 fold increase over the first 4 days (Figure 24A).
Wholemount in situ hybridisation and sectioning of ES and EPL cell embryoid
bodies indicated that SPARC expression was confined to an outer endodermal
cell layer in both ES and EPL cell embryoid bodies at day 4 (Figure 24B,C),
indicative of parietal endoderm formation. AFP levels were too low to be
detected
during these stages by Northern or RNase protection, so wholemount in situ
hybridisation of embryoid bodies was used to detect AFP expression. At day 3,
1 % of ES cell embryoid bodies exhibited discrete patches of AFP expressing
cells
on their surface. This level rose to 52.9 +/- 9.5% of ES cell embryoid bodies
by
day 4 {Figure 24D). Sectioning confirmed that AFP expression was confined to
outer cells and was therefore representative of visceral endoderm (Figure
24F).
AFP expression could not be detected on surface or interior cells of EPL cell
embryoid bodies at day 3 or day 4 of embryoid body development (Figure 24E).
Cell mixing experiments were carried out to determine whether the failure of
EPL cells to form visceral endoderm reflected an inherent restriction in the
developmental potential of these cells or an alteration in the embryoid body
environment. The KSF-4 ES cell line constitutively expresses a LacZ gene
modified to target ~i-galactosidase protein to the nucleus. Analysis of
embryoid
bodies formed from KSF-4 ES cells demonstrated formation of visceral endoderm
at levels comparable with D3 ES cells (data not shown). After passaging in the
absence of mouse embryonic fibroblast feeder cells, KSF-4 ES cells were
cultured
for 2 days in MEDII to form KSF-4 EPL cells (EPL (LZ+)). Embryoid bodies
generated by co-aggregation of a 1:1 ratio of D3 ES cells and EPL {LZ+) cells
were assessed for visceral endoderm fom~ation by AFP expression at day 4. ES
cell embryoid bodies and EPL {LZ+) cell embryoid bodies gave rise to visceral
CA 02324591 2000-09-19
WO 99/53021 PCTIAU99/00265
$8 _
endoderm at levels consistent with those previously described (65% and 0% of
bodies respectively). Embryoid bodies formed from the mixed cell population
gave rise to visceral endodem~ at levels comparable to ES embryoid bodies
(51 %). Double staining for ø-galactosidase activity and AFP expression
revealed
the presence of both LacZ+ and LacZ cells within the visceral endoderm (Figure
25). This suggested that the inability of EPL embryoid bodies to give rise to
visceral endoderm resulted from a deficiency in the embryoid body environment
rather than an inherent developmental restriction of EPL cells, and implies
the
existence of an inductive signal for visceral endoderm formation.
Differentiation of pluripotent cells in the absence of visceral endoderm
The formation of EBs from EPL cells provides a methodology for the
differentiation of pluripotent cells in the absence of visceral endoderm.
Visceral
endoderm is known to express signals that influence pluripotent cell
differentiation
in vivo. Differentiation in the absence of this cell type provides an
opportunity for
specific control of pluripotent cell differentiation by the addition of
factors and
environmental alteration. This cannot be achieved in ES cell EBs in which
pluripotent cell differentiation is at least partly controlled by signals from
visceral
endoderm which differentiate spontaneously from exterior ES cells.
Accelerated and enhanced mesoderm formation in EPL cell EBs
The formation of early mesoderm within ES and EPL cell EBs was
monitored by analysing expression of the early mesodermal markers, brachyury
and goosecoid Consistent with previous reports, brachyury expression was
barely detectable on days 0-3 of developrent in ES cell EBs, but was up
regulated on day 4 (Figure 26). Coosecoid expression could not be detected in
ES cell EBs during the course of this experiment. in contrast, brachyury and
goosecoid expression were up regulated 30 and 6 fold respectively on days 2
and
3 of EPL cell EB development, followed by 9 (brachyury) and 6.5 (goosecoid)
fold
decreases in expression on day 4 {Figure 26). In both ES and EPL cell embryoid
bodies the expression of mesodermal markers immediately preceded decreases
in the expression levels of primitive ectoderm markers FgfS and Oct4 (Figure
23).
CA 02324591 2000-09-19
WO 99153021 PC?/AU99/00265
89
Brachyury expression in ES cell embryoid bodies did not reach the levels seen
in
EPL cell bodies at day 3/4 even after extended culture.
Wholemount in situ hybridisation was used to detect the extent of brachyury
expression within the embryoid body populations. Brachyury expression was
detected in 1 % of ES cell EBs at day 3 and 16% of bodies at day 4 (Figure
26B,D). By contrast, 98% and 92% of EPL cell EBs exhibited brachyury
expression on days 3 and 4 respectively (Figure 26C,E). A similar expression
pattern was observed with goosecofd (data not shown). Oct4 expression within
ES and EPL cell embryoid bodies exhibited the expected inverse correlation
with
the onset and extent of brachyury expression. Oct4 expression was relatively
uniform throughout control EBs on days 3 and 4 (Figure 26F,H), but patchy
within
EPL cell EBs where mesoderm differentiation had commenced (Figure 26G,1).
The comparison of markers specific for nascent mesoderm suggested that EPL
cell embryoid bodies undergo an accelerated differentiation program resulting
in
the earlier appearance and more extensive formation of mesoderm compared to
ES cell embryoid bodies.
Tem~inal differentiation of nascent mesoderm was monitored by the
appearance of beating cardiocytes (Figure 27A). This was first detected in ES
cell
EBs at day 8 of development (8%) and increased with time, reaching 36% of
bodies at day 12. In EPL cell EBs, beating muscle was observed in 14%
embryoid bodies by day 6, two days prior to its appearance in ES cell EBs, and
increased steadily to 60% by day 10 and 12 of ernbryoid body development. The
proportion of EPL cell EBs containing beating muscle was higher than ES cell
EBs
at all time points. Consistent with this profile, expression of NIor2.5 was
induced
earlier in EPL cell EBs, by day 6 compared to day 8 in ES cell EBs (Figure
27B).
Furthermore, while N1a2.5 expression levels increased in both ES cell and EPL
cell EBs to day 12, levels in ES cell EBs were approximately 3 fold below
those
observed in EPL cell EBs throughout this period. The enhanced ability of EPL
cells to form cardiac muscle when differentiated as embryoid bodies presumably
reflects the accelerated and increased formation of nascent mesoderm.
CA 02324591 2000-09-19
WO 99/53021 PGTIAU99/00265
90 -
The fom~ation and differentiation of EPL cells as EBs therefore provides a
methodology for efficient programming of pluripotent cell differentiation into
mesodermal lineages. Mesodermal progenitors are formed earlier and at much
higher levels than in ES cell EBs.
EPL cell embryoid bodies exhibit reduced capacity for neuron formation
Differentiation of EPL cells into ectoderm derived iineages was assessed by
the presence of neurons within individual embryoid bodies (Figure 28A), and
compared to ES cell EBs. Neurons were not detected in either embryoid body
population before day i 0. On day 10, 26% of ES cell EBs contained obvious
neural networks. This rose to 41 % by day 12. Embryoid bodies derived from EPL
cells failed to form neurons.
ES cells and P19 embryonal carcinoma (EC) cells form neurons when
differentiated as aggregates in the presence of retinoic acid (RA). To examine
whether the failure of EPL cell EBs to give rise to neurons resulted from an
inherent restriction in neuron differentiation capacity, the ability of EPL
cells to
differentiate into neurons after aggregation in the presence of RA was
assessed.
Individual RA-treated ES and EPL cell aggregates were scored for the presence
of
neurons (Figure 28B) and found to exhibit similar frequencies of neuron
formation
at 63% and 68%, respectively. This indicated that the absence of neurons
within
EPL cell EBs did not reflect an inherent restriction in the capacity of EPL
cells to
form neurons, but an altered embryoid body environment which resulted in
reduced neural specification.
In summary, EPL cell EBs formed nascent mesoderm and mesoderm
derivatives with high efficiency, but failed to form neurons despite retaining
neuron-forming potential. Differentiation in EPL cell EBs may reflect directed
formation of the mesoderm germ layer from piuripotent cells cultured under
these
conditions. This may result from the demonstrated failure of EPL cell EBs to
form
an outer layer of visceral endoderm which has been demonstrated to ptay a role
in
the specification of ectodermal iineages.
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
91
Differentiation of EPL cells in response to cytokines/grvwth factors
Diifferentiation of ES cells and EPL cells in response to members of the FGF
growth factor family
ES and EPL cells were seeded into DMEM + LIF or DMEM + 50% MEDII +
LIF respectively at a density of at 75 cells/cm2. Cells were cultured in the
presence of increasing concentrations (0.1-100 ng/ml) of recombinant bovine
basic fibroblast growth factor (bFGF; Boehringer Mannheim} for a period of 5
days. Addition of bFGF to ES cells in the presence of L1F had no effect on ES
cell
morphology, differentiation or proliferation. In contrast, addition of 10
nglml or
higher concentrations of bFGF induced the differentiation of EPL cells to a
characteristic cell type (cell type A; Figure 17A) which did not stain for
alkaline
phosphatase. Similar results were obtained with other members of the FGF
family
including human recombinant acidic FGF {R&D Systems; 10 ng/ml), human
recombinant kFGFIFGF4 (Sigma). Human recombinant FGFS (R&D Systems) did
not induce ES or EPL cell differentiation at concentrations up to 500 ng/ml
Individual colonies within representative differentiation cultures induced
with
l0ng/ml bFGF were classed as ES cell, EPL cell, differentiated cell, or mixed
EPUdifferentiated cell on the basis of morphology and alkaline phosphatase
staining (Figure 29B}. The proportion of undifferentiated ES cell colonies was
similar regardless of the presence of bFGF. The addition of bFGF to EPL cell
cultures caused a marked decrease in the level of EPL cell colonies, from 58%
to
21 %, with a corresponding increase in colonies containing differentiation
(42% to
79%), indicating that bFGF was inducing the differentiation of EPL cells but
not ES
cells in culture.
Activin A induces the differentiation of EPL cells but not ES cells.
ES and EPL cells were seeded at a density of 75 cell/cm2. Cells were
cultured for 5 days in DMEM + LIF or DMEM + 50% MEDiI + LIF respectively, to
which had been added increasing concentrations of recombinant human activin A
(1-200 nglml; courtesy of Dr. R. Rogers, Fiinders Medical Centre, S.A.).
Activin A
had no effect on ES cell rnorphofogy, differentiation or proliferation at any
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
92
concentration tested. Addition of activin A to EPL cells in culture induced
their
differentiation. After 5 days the majority of colonies in activin A treated
wells had a
distinctive colony morphology (Figure 29C) characterised by the presence of a
fibroblastic cell type which did not stain for alkaline phosphatase activity.
This cell
type was morphologically distinct from cell type A, formed by differentiation
of EPL
cells with bFGF, and is referred to as cell type B.
Individual colonies within representative differentiation cultures induced
with
150ng/ml activin A were classed as ES cell, EPL cell, differentiated cell, or
mixed
EPUdifferentiated cell on the basis of morphology and alkaline phosphatase
staining (Figure 29D). The proportion of undifferentiated ES cell colonies was
similar regardless of the presence of activin A. The addition of activin A to
EPL
cell cultures resulted in efficient cell differentiation as assessed by a
marked
decrease in the proportion of EPL cell colonies from 52% to 3%, and an
increase
in the proportion of colonies containing differentiated cells from 48% to 97%:
Reverted EPL cells regain ES cell differentiation capability
EPL cells revert to ES cells when cultured in the absence of MEDII but in
the presence of LIF as assessed by morphology, gene expression, cytokine
responsiveness and ability to contribute to chimaeras following blastocyst
injection
(Example 2). ES cells were differentiated to EPL cells by culture in MEDII for
2
days, before passaging into medium containing LiF to form reverted EPL cells
(EPLR). Embryoid bodies were formed from each ES, EPL and EPLR cell
population and their differentiation was compared.
Embryoid bodies derived from EPLR cells were identical in morphology to
ES cell EBs and could be distinguished easily at day 4 from EPL cell EBs
(Figure
22; data not shown). Expression of brachyury in ES and EPL cell EBs (Figure
30A) was consistent with the profile previously described (Figure 26).
Brachyury
expression in EPLR cell EBs was similar to that observed for ES cell EBs, with
expression detected at low levels on day 4 of deveioprnent and not at high
levels
on day 2 and 3 as described for EPL cells. Compared to EPL EBs, which failed
to
give rise to visceral endoderm, EPLR EBs gave rise to visceral endoderm at
levels
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
93 -
comparable to ES EBs (31 % and 46% respectively), as shown by whofemount in
situ hybridisation. Microscopic analysis of individually seeded ES, EPL and
EPLR
EBs (Figure 30B,C) indicated that reversion of EPL to EPLR cells was
accompanied by restoration of high levels of neuron formation and reduced
levels
and later appearance of beating cardiocytes. Reverted EPL cells therefore have
a
similar differentiation capacity to ES cells both in vitro and in vivo
(Example 2).
Summary
Comparative analysis of ES and EPL differentiation confirmed the
pluripotent nature of EPL cells, supported the identification of EPL cells as
primitive ectodem~-like, and defined ES and EPL cells as developmentally
distinct
populations on the basis of aftemate differentiation capabilities and growth
factor
responsiveness. In combination with Example 2, this indicates that ES and EPL
cells have distinct differentiation capacities both in vitro and in vivo.
The products of ES and EPL cell differentiation within embryoid bodies
differ both in the proportion of ectodermal and rnesodermal germ layers and
derivatives, and in the formation of the extraembryonic lineage visceral
endoderm.
EPL cell embryoid body formation therefore provides:
- a methodology for the differentiation of pluripotent cells
in the absence of visceral endoderm and the inductive signals produced
by visceral endoderm. This provides unprecedented opportunity for
control of piuripotent cell differentiation including directed lineage
specific differentiation.
- a methodology for efficient programming of pluripotent
cell differentiation to a mesodemlal fate by differentiation to EPL cells
followed by formation of embryoid bodies or treatment with mesoderm-
inducing growth factorslcytokines.
CA 02324591 2000-09-19
WO 99/53(121 PCTIAU99/00265
94 -
EXAMPLE 7
Alternative formation of ectoderm and mesoderm germ layers and
derivatives by directed differentiation of pluripotent cells in vitro
The use of factors within MEDII has enabled the development of a strategy
for the production of a uniform population of pluripotent EPL cells in vitro,
either as
colonies in adherent culture or as aggregates in suspension, representative of
primitive ectoderm. Further, the failure of EPL cell embryoid bodies to form
the
instructive cell type visceral endoderm, and the responsiveness of EPL cells
to
inductive signals that do not differentiate ES cells provides an opportunity
for
direct control of piuripotent cell differentiation.
In this example we demonstrate that the differentiation of pluripotent EPL
cells can be controlled in vitro to generate cell types representative of
specific
embryonic germ .layers, notably ectoderm and mesoderm. These germ layer cells
can be used for the generation of differentiated derivatives with potential
for
commercial, medical and agricultural use. Specifically, manipulation of
environmental stimuli, such as alteration of the extracellular matrix and/or
the
addition of exogenous soluble factors, can be used for efficient and
programmed
formation of alternative germ layers. Further manipulation of the environment
will
enable the directed formation of individual cell iineages from the gems
layers,
allowing the production of specified cell types and differentiation
intermediates.
Examples of this strategy are outlined below.
Materials and Methods
Cell culture conditions
All cells and tissue culture techniques were as described in Examples i and
6 unless otherwise stated.
Lineage specific differentiation: formation of neurectoderm
For the production of neurectoderm, EBMs (Example 1 ) were collected on
day 4 and resuspended in DMEM + 50% MEDII with or without 20 ng/ml FGF4
CA 02324591 2000-09-19
WO 99I530Z1 PCT1AU99/00265
95 -
(Sigma) in bacterial petri dishes and maintained for a further 3 days.
Cellular aggregates used for the analysis of gene expression by in situ
hybridisation were subsequently seeded for 16 hours (day 8) onto tissue
culture
plates pre-coated with gelatin prior to fixing with 4% PFA. Control aggregates
were ES cell EBs formed from ES cells in DMEM in the absence of MEDII.
Cellular aggregates used for the analysis of gene expression at timepoints
after
day 8 by in situ hybridisation were seeded onto gelatinised tissue culture
plastic in
50:50 DMEM:F12 (F12, Gibco BRL) + 10% FCS. After 1fi hours the medium was
changed to 50:50 DMEM:F12 supplemented viiith 1TSS (Boehringer Mannheim), 1
mM L-glutamine and 10-20 nglrnl recombinant human FGF4. Aggregates were
maintained in this medium for 1-4 days prior to fixing with 4% PFA.
For functional differentiation assays to assess the ability of neurectoderm
formed from ES cells to diffet'entiate into neurons, aggregates on day 7 were
seeded individually into 2 ml tissue culture wells and maintained as described
above. The formation of neurons was assessed on days 8, 10 and 12.
Lineage specific differentiation: formation of mesoderm
Formation of cells of mesodermal lineage has been achieved using several
similar approaches.
Prom EPL cells generated in adherent culture: Mesoderm was formed
from EPL cells formed by adherent culture in the presence of MEDII as
described
in Example 1. These cells were trypsinised to a single cell suspension and
seeded at a density of 1 x 105 celis/ml into bacterial plates to form EPL cell
EBs as
described in Example 6.
From EPL cells formed in suspension (EBMs): EPL cells, formed as cell
aggregates (EBMs) in suspension culture, were differentiated into mesoderm by
trypsinisation to a single cell suspension and reaggregation in bacterial
dishes in
DMEM or DMEM + 7 0 ng/ml FGF4. Cells were seeded at a density of 1 x 105
CA 02324591 2000-09-19
WO 99153021 PCTIAU99/OOZ65
96 -
cells/ml.
Macrophage formation from ES and EPL cells
On day 0, ES and EPL cells were trypsinised and seeded in DMEM into
bacterial grade petri dishes at a density of 1 x1 OS celUml to allow aggregate
formation. On day 2, approximately 100 aggregates from each plate were
collected and seeded into 1.25m1 MC media (0.9% methyl cellulose in Iscoves
modified Dulbecco's medium (IMDM), 15% FCS, 50 mg/ml ascorbic acid, and
4.5x10'~M monothioglycerol (MTG)) supplemented with 400 Ulml IL-3 (courtesy of
Dr. T. Gonda, IMVS, Adelaide) and 10 ng/ml recombinant human M-CSF (R&D
Systems). On Day 14 and Day 18, 50 colonies of each cell type were scored as
containing 5 or more macrophages (positive) or less than 5 macrophages
-- {negative).
Cells formed in MC cutture were collected by centrifugation, cytospun, and
air dried. Macrophages were identified by morphology when stained with
May-Grunwald-Giemsa stain, and by positive staining with the
macrophage-specific antibody F4/B0 (Austyn and Gordon, 1981 ).
Gene expression analysis
Gene expression analysis by in situ hybridisation, northern blot and RNase
protection were performed as described in Examples 1 and 6. Additional probes
were: Sox1 riboprobes for wholemount in situ hybridisation were generated from
plasmid #1022 (obtained from Dr. Robin Lovell-Badge, NIMR, London) linearised
with BamHl and transcribed with T3 RNA polymerase (anti-sense) or linearised
with Hindlll and transcribed with T7 RNA polymerase (sense). Antisense Sox7
probes for RNase protections were generated from a plasmid containing a -450
by XhollEcoR1 fragment from the Sox1 cDNA in pfasmid #1022 subcloned into
pBluescript ll KS+. The resulting plasmid was linearised with Kpn1 and
transcribed with T7 RNA polymerase. Sox2 probes for use in in situ
hybridisation
were generated from plasmid #1015 (obtained from Dr. Robin Lovell-Badge,
NIMR, London) finearised with Accl and transcribed with T3 RNA pofymerase
CA 02324591 2000-09-19
WO 99153021 PCT/AU99100265
97 -
(antisense) or finearised with Noti and transcribed with T7 RNA polymerase
(sense). An anti-sense Gbx2 riboprobe for use in in situ hybridisation were
generated from pGbx2c7.1 (Chapman et al., 7 997) linearised with Alul and
transcribed with T3 RNA poiymerase.
immunohistochemical Analysis
Seeded cellular aggregates were fixed in 4% paraformaldehyde in PBS and
blocked in the appropriate blocking buffer for 30 minutes followed by
overnight
incubation with primary antibodies in blocking buffer at 4°C.
Aggregates were
then rinsed with species specific secondary antibodies in blocking buffer for
1
hour. For FITC-conjugated secondary antibodies, propyl gallate 5 mg/ml was
added. Slides were examined on a Nikon TE300 microscope using fluorescence.
. Nestin: Blocking buffer: 1 % goat serum, 1 mgJml BSA, 1 % Triton X 100 in
PBS. Primary antibody: Developmental Studies Hybridoma Bank, reference Rat
401, used at a dilution of 1:100. Secondary antibody: FITC conjugated goat
anti-
mouse IgM (~-specific: Sigma) used at a concentration of 1:100.
N-Cam: Blocking buffer: 1 % FCS, 1 mg/ml BSA, 1 % Triton X 100 in PBS.
Primary antibody: Santa Cruz Biotech, SC-1507 used at a concentration of 1:20.
Secondary antibody: FITC conjugated rabbit anti-goat IgG (Sigma) used at a
ditution of 1:700.
NF200: NF200 was detected by indirect immunofluorescence as described
by Robertson (1987). Primary antibody: anti-neurofilament 200 {Sigma
immunochemicals N-4142) used at a dilution of 1:200. Secondary antibody:
FITC-conjugated anti-rabbit igG (Silenus) used at a dilution of 1:60.
Results
Programmed formation of ectodermal, neurectodermal and neural iineages
by pluripotent cell differentiation in vitro
EBMs, formed by aggregating ES cells in DMEM + 50% MEDII for 4 days in
CA 02324591 2000-09-19
WO 99153021 PCTIAU99/00265
98
suspension culture (Example 1 ), were maintained in DMEM + 50% MED11, or
DMEM + 50% MEDII + 20 ng/ml FGF4 for a further 3 days in suspension. Under
these conditions approaching 100% of EBMs developed into cellular aggregates
with a distinctive layered structure of pseudo-stratified epithelial
appearance
(Figure 31 A). These layers were observed in aggregates cultured in 50% MEDII
alone but were enhanced in the presence of FGF4. However, these layers were
never seen in EPL cell EBs, and were observed only sporadically in ES cell EBs
where they comprised a subset of cells within the body.
The identity of cells within these iayers was tested by analysis of the
expression of transcripts and cell surface. markers diagnostic for embryonic
cell
types. For the analysis of gene expression by in situ hybridisation,
aggregates
were seeded after 7 days of suspension culture for 16 hours onto gelatin
treated
tissue culture plastic in DMEM + 50% MEDII +l- FGF4 (Figure 31 B). Expression
of the pluripotent cell marker Ocf4 could not be detected in the cell layers
indicating that these cells were not pluripotent. Brachyury expression was not
detected by in situ hybridisation, demonstrating that these structures did not
contain nascent mesodeml. Further, no cell types of mesodermal morphology
could be recognised within the aggregates.
Sox) is first expressed by the neurectoderm/neural plate in vivo and by all
undifferentiated neural cells. it is not expressed in cell types of mesodem~al
or
endodemlal origin. Gbx2 is expressed in the primitive streak, then in the
neurectoderm prior to neural tube closure and subsequently at the
midbrain/hindbrain boundary. Sox2 is expressed in the neurectoderm after
neural
tube closure and persists until terminal differentiation of neural cells. In
situ
hybridisation demonstrated widespread expression of Sox1 by cells within the
layers, and Gbx2 by a subset of these cells. This suggested that the cell type
produced by these culture conditions was equivalent to neurectoderm,
specifically
early neural plate around the time of neural tube closure.
The analysis of neural gene expression within these aggregates was
refined by analysis of neural specific markers by in situ hybridisation {Soxl,
Sox2
and Gbx2; Figure 32A) and immunohistochemistry (nestin and N-CAM; Figure
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99I00265
99 -
32B) on days 8, 9 and 10. Sox1 expression was detected in greater than 90% of
aggregates on day 8 of development, however, expression was seen in only a
proportion of cells in each aggregate. Sox1 expression increased on day 9 and
of development, with approaching 100% of cells within each aggregate
5 expressing Sox7. Similarly, on day 10 the majority of cells were also
expressing
Sox2. Gbx2 expression was observed in a proportion of cells on day 8 of
development and was down-regulated on days 9 and 10. This expression pattern
is consistent with identification of the cell layers as neurectodermal in
origin.
The identification of these cells as neurectoderm was supported by the
10 expression of neural proteins (Figure 32B, data not shown). Nestin, an
intermediate filament protein expressed by undifferentiated neural stem cells,
was
expressed in the cell layers at days 8, 9 and 10, similar to SoxT. N-Cam is a
marker expressed by all neural lineages, including both undifferentiated and
differentiated cells. N-Cam staining was detected throughout the~aggregates on
days 8, 9, and 10, with the number of cells expressing N-Cam increasing to
nearly
100%. N-Cam was expressed both in the cell layers and in differentiated cells
surrounding the tayers, indicating that the majority of the differentiated
cells were
of neural origin.
Northern blot and RNase protection analysis of gene expression supported
the conclusion that EBMs were programmed to form neurectoderm in the
presence of MEDII. Oct4 and Fgf5 expression (Figure 33A) were markedly down
regulated in EBM aggregates between day 4 and day 5, indicating the loss of
pluripotent cells. However, a low but consistent level of Ocr4 transcript was
detected in EBM aggregates after day 5. In situ hybridisation (Figure 31 B)
demonstrated that the low Oct4 transcription was not expressed from residual
pluripotent cells within the aggregate and may be a feature of the early
neurectoderm in these aggregates. This residual expression could not be
detected in ES cell EBs which form low levels of neurectoderm. Expression of
brachyury was observed from day 4 in ES cell EBs, consistent with the loss of
Oct4 expression, but could not be detected in EBMs (Figure 8). Finally, Gbx2
and
Soxl expression (Figure 33B) were detected by RNase protection in aggregates
CA 02324591 2000-09-19
WO 99153021 PCTIAU99I00265
100 -
formed from EBMs on days 6, 7 and 8. While expression of Gbx2 decreased on
day 8, Soxt expression continued to increase to day 10.
These results demonstrate that pluripotent cells can be programmed
specifically to an ectodermal and neurectodermal fate by factors within MEDII.
The ectodermal cells are formed in the absence of mesodermal cell types, and
exhibit a temporal pattern of gene expression equivalent to neurectoderm in
vivo,
with progression from an early neurectodermal cell type equivalent to neural
plate,
to a later neurectodermal cell type present after neural tube closure. In the
embryo these cells are precursors for all neural Iineages.
Neurectoderm derived by directed pluripotent cell differentiation in vitro can
be further differentiated to neural cell types.
Individual aggregates developed from EBMs in the presence of MEDII, or
ES cell EBs, were seeded and assessed on days 8, 10 and 12 for the presence of
neurons, identified morphologically by the presence of axonal projections (and
i5 confirmed by~the expression of NF200; data not shown), and beating
cardiocytes.
Beating cardiocytes (Figure 34A) could be detected in over 50% of ES cell EBs
on
day 8, and these persisted through days 10 and 12 of development. In contrast,
the vast majority of EBM-derived aggregates (99.6%) maintained in MEDII did
not
contain beating cardiocytes. The lack of beating cardiocytes, a mesoderrnally
derived tissue, in these aggregates, is consistent with the lack of brachyury
expression at earlier stages of development (Example 1, Figure 7, 8) and
indicates that mesodermal lineages are not formed in these aggregates. Neurons
were not detected in either population of aggregates on day 8 of development
(Figure 34B), but were apparent in over 60% of EBM-derived aggregates on day
10. By day 12, in excess of 90% of these aggregates contained neurons. By
comparison, formation of neurons was observed in 10% (day 10) and 25% (day
12) of ES cell EBs. These data support the earlier conclusion that
neurectoderm
is formed at high levels and in the absence of mesodermaf cells by EBMs
cultured
in the presence of MEDII, and demonstrate that this neurectoderm can give rise
to
terminally differentiated neural cell types. Aggregates developed from EBMs in
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99I00265
101 -
MEDII are therefore enriched in undifferentiated neural cells.
Neurectoderm formation in vivo proceeds via the formation of definitive
ectoderm. Although no markers exist for definitive ectoderm in vivo, gene
expression analysis identified a population of cells in EBMs programmed to
form
neurectoderm which expressed low levels of Oct4 and failed to express
neurectodermal markers. These cells were present transiently between primitive
ectoderm (high Oct4 expression) and neurectoderm (high Soxl, Gbx~ and
suggest that neurectoderm formation proceeds via an intermediate population
which may represent definitive ectoderm.
The formation of neurectoderm described here does not rely on the addition
of chemicai inducers, such as retinoic acid, or genetic manipulation to
promote
neural formation. Instead, it relies on biologically derived factors found
within the
conditioned medium MEDII. Neural progenitors formed in this manner are thought
to be differentiated from pluripotent cells in a manner analogous to the
formation
of neural cells during embryogenesis and are therefore ideal for the
production of
differentiated neural cells useful for commercial, medical and agricultural
applications. Further, in contrast to the formation of limited neural iineages
by
chemical inducers such as retinoic acid, the identity of neural cell types
produced
using these methodologies is not likely to be restricted.
Neurectoderm programming of pluripotent cell differentiation requires the
large molecular weight components, but not the small molecular weight
components, of MEDII.
During embryogenesis neurectoderm forms from anterior primitive
ectoderm cells that maintain association with the basement membrane/
extracellular matrix. Identification of a bioactive ECM protein within the
large
molecular weight component of MEDII prompted an investigation of the role of
this
activity in directed neurectoderm formation from pluripotent cells. This was
tested
by the ability of this component to induce formation of neurons within EPL EBs
in
which neural cell types are not normally formed (Example 6).
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
102 -
EPL cells were formed by culturing ES cells for 2 days in the presence of
MEDII and in the absence of LIF. EPL cell EBs were formed in DMEM or DMEM
+ R (Example 4). On day 4 of development embryoid bodies were individually
seeded into gelatin treated 2 ml tissue culture wells. Beating cardiocytes and
neurons were assessed on days 7, 8, 9 and 10 of development (Figure 35).
EPL cell EBs formed beating muscle with high efficiency in the absence of
the large molecular weight component and none of these EBs contained neurons.
Inclusion of the large molecular weight component (50-100 Nglml) during
embryoid
body formation resulted in the development of neural cells in about 5% of the
EBs
at day 9, and in 10-12% at d 10 and 72. Only 10-i2% of EPL cell EBs formed in
the presence of the large molecular weight component contained beating muscle,
and the formation of beating muscle was delayed until day 9. These experiments
were carried out in the presence of 1 uglml neutralising anti-human LiF
antibody
(R&D Systems). The altered developmental program could therefore not be
attributed to low levels of LIF expressed by the Hep G2 cells.
This experiment demonstrates a role for the large molecular weight
component of MEDII, and possibly ECM components, in programming of
differentiation within pluripotent cell aggregates so as to delay and reduce
mesoderm formation, and promote the formation of neurons.
Efficient programmed formation of mesodermal lineages from pluripotent
cells
Pluripotent cells can be programmed to adopt a specific mesodermal fate
by differentiation through a homogeneous EPL cell intermediate as shown in
Example fi. This may reflect the absence of visceral endoderm within EPL cell
EBs. Nascent mesoderm formation can be detected in approaching 100% of EPL
cell EBs and is probably underestimated by scoring beating cardiocytes which
comprise only one of the possible nascent mesoderm cell fates. The previous
experiments were carried out using EPL cells formed from ES cells in adherent
culture. Here we describe alternative methodologies for efficient mesoderm
formation from pluripotent cells in vitro.
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
103 -
Formation of mesoderm by reaggregation of EPL cells formed in suspension
EPL cells formed in suspension in the presence of MEDlI (EBMs, Example
1 ) comprise a stable and homogeneous population of cells with properties
equivalent to embryonic primitive ectoderm. Programming the differentiation of
these cells to a mesodermal fate is thought to require both removal of the
neurectoderm-inducing activity of MEDfI and dissociation from ECM components.
This would mimic the behaviour of pluripotent cells destined to form mesoderm
during primitive streak formation in vivo.
On day 4 EBMs were trypsinised to a single cell suspension and
reaggregated in DMEM +I- 10 ng/ml FGF4, or DMEM + 50% MEDII +I- 10 nglml
FGF4. After a subsequent 4 days of development an obvious morphological
d'rfference was apparent between reaggregates cultured in DMEM and those
cultured in DMEM + 50% MEDI( (Figure 36). RNA from these aggregates on days
2 and 4 was analysed by northern blot for expression of brachyury and Oct4
(Figure 36). Oct4 expression was down regulated in reaggregates on day 4,
indicating the -loss of pluripotence as differentiation occurred. Brachyury
expression could not be detected in reaggregates developed in DMEM + 50%
MED11, consistent with the fact that these are destined to form neurectoderm.
Brachyury expression, indicating mesoderm formation, was strongly up regulated
in reaggregates developed in DMEM at day 2 and 4 in the absence of FGF4. fn
the presence of FGF4 brachyury expression peaked on day 2 and was down
regulated by day 4, suggesting that addition of FGF4 resulted in accelerated
mesodermal differentiation of the pluripotent cells. The expression of marker
genes was confirmed by in situ hybridisation of reaggregated bodies with Oct4
and
brachyury specific probes (data not shown). These data indicate that EPL cells
formed in suspension can be programmed to differentiate into mesoderm.
Mesodermal precursors derived by directed pluripotent cell differentiation
can be programmed to alternative developmental fates
Programmed formation of high levels of mesodem~~vn EPL cell EBs was
demonstrated by formation of nascent mesoderm and beating cardiocytes
(Example 6). To establish whether the elevated levels of nascent mesoderm in
CA 02324591 2000-09-19
WO 99153021 PCTIAU99/00265
104 -
EPL cell EBs were developmentally restricted to myogenic lineages or could be
programmed to alternative developmental fates, the formation of macrophages in
response to exogenous cytokines was assessed.
EPL cell and control ES cell EBs were differentiated in MC media
supplemented with mIL-3 and hM-CSF, and scored for the presence of
macrophages on days 12, 15 and 18 (Figure 37). On day 12, 32.4% of EPL cell
EBs were observed to contain macrophages, compared to 4.8% of ES cell EBs.
On days 15 and 18 the proportion of embryoid bodies containing macrophages
had increased to 43% of EPL cell EBs and 9-10 % of ES cell EBs. Consistent
with the eariier formation of mesoderm in EPL cell EBs, formation of
macrophages
initiated approximately two days earlier in these embryoid bodies compared to
ES
cell EBs (data not shown). Enhanced formation of multiple mesodermal lineages
in EPL cell EBs suggests that the elevated nascent mesoderm in these bodies
contains a multipotent mesodermal progenitor which can be programmed to
alternative fates in response to specific environmental cues.
Pluripotent cell differentiation can be specffically programmed to the
formation of alternative mesodermal and ectodermai lineages
A comparative analysis of methodologies devetoped for directed
mesodermal (formation of EPL cell EBs) and ectodem~al (formation of EBMs)
differentiation of pluripotent cells is presented in figure 38. Differentiated
aggregates were scored for the presence of neurons and beating muscle as
representative cell types for mesodermal and ectoderma! lineages respectively.
EPL cell EBs formed beating muscle with high efficiency but were unable to
form neurons. EBMs showed the reciprocal pattern, forming neurons with high
efficiency but failing to form beating muscle. The formation of mesoderm in
particular is probably underestimated in this analysis because only a single
mesodermal cell type, beating muscle, was scored. Appropriate use of the
factors
within MEDII to form and differentiate pluripotent cell populations can
therefore be
used to direct specific formation of alternative mesoderm and ectoderm germ
layers from pluripotent cells in vitro.
CA 02324591 2000-09-19
WO 99/53021 PCT'/AU99/00265
105
Summary
We have described methods for directing the differentiation of pluripotent
cells into cell Iineages of alternative germ layers, namely ectoderm and
mesoderm. Formation of EPL cells in response to the biological activity in
MEDII,
in adherent or suspension cultures, is an essential step in these lineage
specific
differentiation protocols. Although the pluripotent cells used in the above
examples were obtained by culture of ES cells in MEDII, we envisage that
primary
pluripotent cells isolated from embryonic ICM, primitive ectoderm or PGCs, or
pluripotent cells obtained by dedifferentiation or nuclear transfer, could be
used as
a source of material for controlled lineage specific differentiation in vitro.
Neurectoderm formation, in the absence of mesoderm formation, occurred
when EPL cells were differentiated in the presence of MEDII in suspension
culture. This suggested that the conditioned medium contained components
required for neural determination. Fractionation of the conditioned medium
suggested that this component resided in a fraction containing medium
components of greater than 10 kDa, and may be the ECM component identified
as active in the formation of EPL cells. Formation of neurectoderm from
primitive
ectoderm in contact with ECM components is consistent with the determination
of
this Lineage during embryogenesis.
Efficient formation of mesoderm from EPL cells, in the absence of
neurectoderm, could be achieved in adherent or suspension culture and required
removal of MEDII, potentially to eliminate contact with ECM proteins. This
could
be achieved by aggregating, or reaggregating, EPL cells in medium without the
addition of MEDII, or seeding EPL cells onto gelatin treated plastic-ware in
the
presence of members of the TGF~i or FGF families. Differentiation of EPL cells
as
cellular aggregates proceeded in the absence of visceral endoderm and resulted
in the fom~ation of mesodermal cell lineages, as assessed by gene expression
and terminal differentiation.
Importantly EPL cells, and the inclusion or withdrawal Qf MED11 or MEDII
activity, provide an approach for the production cell populations highly
enriched in
CA 02324591 2000-09-19
WO 99/53021 PC?/AU99/00265
106 -
progenitor and differentiated cells derived from a single germ layer. This
approach does not involve the addition of chemical inducers (such as retinoic
acid
for neural cell formation) or prior genetic modification of the pluripotent
cells.
Instead it relies on the factors thought to be involved in neurectoderm and
mesoderm formation during normal embryonic development in vivo. Progenitors
derived in this manner are ideal for projected applications in human medicine,
veterinary applications and agriculture.
EXAMPLE 8:
Gene Therapy Applications
The utility of the present invention in gene therapy is as follows. I depends
on using nuclear transfer technology that has already been developed and used
in
several animal species, including sheep, cattle and mice. A donor human egg is
fertilised in vitro, but then the nucleus removed and replaced by a nucleus
from a
cell from the recipient of the intended gene therapy. The nuclear transferred
egg
is allowed to develop into an early embryo in vitro, and then cells from it
are used
to generate, using the procedures and products of this invention, an EPL or ES
cell fine. This cell line is then genetically modified, using DNA transfer
technology
known to those skilled in the art, to correct a genetic defect, or to replace
or add a
functioning copy of a desired gene; (examples; clotting factor genes for
hemophelia, the CF gene for cystic fibrosis, the insulin gene for Type 1
diabetes).
The modified ES or EPL cell line is then differentiated in vitro to a desired
cell,
tissue or organ, which is reintroduced into the recipient in a therapeutic
transplantation procedure. Since all the genes of the modified cell line (with
the
exception of the modification or the introduced gene) are identical to the
recipient
individual's own genes, there is unlikely to be cell or tissue rejection,
since this
would be in effect, a perfectly matched transplant.
Alternatively, the modified cells from a non-matched human ES or EPL cell
line are genetically modified and then encapsulated, by cell encapsulation
techniques known to those skilled in the art. They are then introduced to a
recipient.as an implant to produce in that individual, a gene product {e.g.
clotting
CA 02324591 2000-09-19
WO 99153021 PCTlAU99I00265
107 -
factor, insulin, etc.,) missing or defective in the treated individual. It is
also
possible to genetically modify a donor established ES or EPL cell line to
render it
non-immunologic to any recipient, thereby generating a "universal ES or EPL
cell
fine". This cell line is further modified to use in the therapeutic procedures
described above, without the use of encapsulation technology.
Such "universal" donor cells are used to generate human endothelial cells,
that are used in xenotranspiantation procedures, for example, to coat
xenotransptant organ blood vessels; e.g. pig kidneys or hearts, with human
endothelium, to reduce or eliminate delayed graft rejection.
EXAMPLE 9
Transplantation of neural cells derived from pluripotent cells.
Despite the promises offered by neural cell transplantation as a long-term
therapy and cure for neurological disorders, significant practical hurdles
remain.
Allogeneic and xenogeneic transplants are still subject to rejection even
though
immunologicai protection is afforded by the blood brain barrier. Furthermore
the
availability of foetal cells, often used in neural transplantation trials, is
scarce.
Neural cells, including neural stem cells developed by controlled
differentiation in
vitro of pluripotent cells along defined neural pathways offers an ideal
source of
cells for neural cell therapy and neural transplantation.
In man transplantation of neural dopamine cells has been trialed to relieve
some of the symptoms of Parkinson's disease, and neural cell transplantation
may
also offer benefits to neurodegenerative disorders. As a first step in
providing
these cells for human therapy, transplantation witf~ neural cells derived from
pluripotent cells can be investigated in a mouse model. Differentiation of ES
cell
lines expressing (i-gaiactosidase directed to a nuclear location (KSF4 cell
line) or
to the cytoplasm (ROSA 26 cell line) can be directed along the neural pathway
using MEDII as described herein. Neurectoderm or differentiated neural cells
can
be injected into cerebral vesicles of neonates. Survival of the transplanted
cells is
CA 02324591 2000-09-19
WO 99153021 PCTIAU99100Z65
108 -
indicated by the maintenance of cells expressing (3-galactosidase in the
brain. The
ability of transplanted cells to integrate can be determined by investigating
the
connections established by marker cells expressing the cytoplasmic form of
~-galactosidase (ie cells derived from the ROSA 26 ES cell line).
References
Abbondanzo, S.J., Gadi, I. and Stewart, C.L. (1993). Derivation of
Embryonic Stem Cell Lines. In: Guide to Techniques in Mouse Development. Eds;
Wassemlan, P.M. and DePhamphilis. Meths. Enzymol. 225, 803-823.
Austyn, J.M. & Gordon (1981 ) F4180, a monoclonal antibody directed
i0 specifically against the mouse macrophage. Eur J. Immunol. 11: 805-815
Bastian, H. and Gruss, P. (1990). An even-skipped homologue, Evx-1, is
expressed during early embryogenesis and neurogenesis in a biphasic manner.
EMBO J. 9, 1939-1952
Berger et al., (1995) The development of hematopoietic cells is biased in
embryonic stem cell chimeras Dev. Biol. 170: 651-663.
Bium, M., Gaunt, SA., Cho, K.W.Y., Steinbeisser, H., Blumberg, B., Bittner,
D. and De Robertis E. M. (1992). Gastrulation in the mouse: the role of the
homeobox gene goosecoid. Cell 69, 1097-1106.
Bradley, A. (1987). Production and analysis of chimaeric mice. in
Teratocarcinomas and embryonic stem cells: a practical approach (ed.
Robertson,
E. J). pp 113-152. IRL press, Oxford.
Bulfone, A., Puelles, L., Porteus, M. H., Frohman, M.A., Martin, G. R. and
Rubenstein, J. L., (1993). Spatially restricted expression of Dlx-1, Dlx-2
(Tes-1 ),
Gbx-2, and Wnt-3 in the embryonic day 12.5 mouse forebrain defines potential
transverse and longitudinal segmental boundaries. J. Neurosci. 7, 3155-3172.
Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
109
isolation by acid guanidinium thiocynate-phenol-chloroform extraction. Anal.
Biochem. 162, 156-159.
Doetschman, T. C., Eistetter, H., Katz, M., Schmidt, W. and Kemler, R.
(1985). The in vitro development of biastocyst-derived embryonic stem cell
lines:
formation of visceral yolk sac, bloood islands and myocardium. J. Embryol.
exp.
Morph. 87, 27-45.
Dush M. K. and Martin, G. R. (1992). Analysis of Mouse Evx Genes: Evx-1
Disptays Graded Expression in the Primitive Streak. Dev. Biol. 151, 273-287.
Dziadek, M. and Adamson, E. (1978). Localization and synthesis of
alphafetoprotein in post-implantation mouse embryos. J. Embryol. Exp Morph.
43,
289-313.
Edwards, D. R., Parfett, C. L. J. and Denhardt, D. (1985). Transcriptional
regulation of two serum-induced RNAs in mouse fibrobtasts: equivalence of
species B2 repetitive elements. Mol. Cell. Biol. 5,3280-3288.
Giger, U. and Meyer, U.A. (1981 ) induction of delta-aminolevufinate
synthase and cytochrome P-450 hemoproteins in hepatocyte culture. Effect of
glucose and hormones. J. Biol. Chem. 256, 11182-11190.
Hahnel, A.C., Rappolee, D.A., Millan, J.L., Manes, T., Ziomek, C.A.,
Theodosiou, N.G., Werb, Z., Pederson, R.A, and Schuttz, G.A. (1990) Two
alkaline phosphatase genes are expressed during early development in the
mouse embryo. Development 110, 555-564.
Haub, 0. and Goldfarb, M. (1991 ). Expression of the fibroblast growth
factor-5 gene in the mouse embryo. Development 112, 397-406.
Hebert, J. M., Basiliico, C., Goldfarb, M., Haub, 0. and Martin G. R. (1990).
Isolation of cDNAs encoding four mouse FGF family members and
characterisation of their expression patterns during embryogenesis. Dev. Blot.
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
110
138, 45~+-463.
Hebert, J. M., Boyle, M. and Martin, G.R. (1991 ). mRNA localisation studies
suggest that the murine FGF-5 ptays a role in gastrulation. Development 112,
407-415.
Herrmann, B. G. (1991 ). Expression pattern of the Brachyury gene in
whole-mount TwisITwis mutant embryos. Development 113, 913-917.
Heukeshoven, J. and Derrick, R. {1985). Characterisation of a solvent
system for separation of water-insoluble poliovirus proteins by reversed-phase
high-pertormance liquid chromatography. J. Chromatogr. 326, 91-101.
Hooper, M., Hardy, K, Handyside, A., Hunter, S. and Monk, M. (1987).
HPRT deficient (Lesch-Nyhan) mouse embryos derived from gems-line
colonization by cultured cells. Nature 326, 292-295.
Hosier, B. A., LaRosa, G. J., Grippo, J. F. and Gudas, L. (1989).
Expression of Rex1, a gene containing zinc finger motifs, is rapidly reduced
by
retinoic acid in F9 teratocarcinoma cells. Mol. Cell Biol. 9, 5623-5629.
Knowles, B.B., Pan, S., Softer, D., Linnerbach, A., Croce, C., Huebner, K.
(1980). Expression of H-2, laminin and SV40 T and TASA on differentiation of
transformed murine teratocarcinorna cells. Nature. 288, 615-618.
Koshimizu, LL, Taga, T., Watanabe, M., Saito, M., Shirayoshl, Y.,
Kishimoto, T. M. and Nalkatsufl, N. (1996). Functional requirement for
gp130-mediated signaling for growth and survival of mouse primordial gems
cells
in vitro and derivation of embryonic germ (EG) cells. Development 122,1235-
1242.
Krieg, P.A. and Melton, D.A. (1987). fn vitro RNA synthesis with SP6 RNA
polymerase. Meth. Enzymol. 155, 397-415.
Laemmli, U.K. {1970) Cleavage of structural proteins during the assembly
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
111 _
of the head of bacteriophage T4. Nature. 227, 680-685.
Lehman. J.M., Speers, W.C., Swartzendruber, D.E. and Pierce, G.B. (1974)
Neoplastic differentiation: charcteristics of cell lines derived from marine
teratocarcinoma. J. Cell. Physiol. 84, 13-28.
Lints, TA., Parsons, L.M., Hartley, L., Lyons, Il., and Harvey, R.P. {1993).
Nkx 2.5: a novel marine homeobox gene expressed in early heart progenitor
cells
and their myogenic descendants. Devetopment 119, 419-431.
Mason, LJ., Taylor, A., Williams, J.G., Sage, H. and Hogan, B.L.M. (1986).
Evidence from molecular cloning that SPARC, a major product of mouse parietal
endoderm, is related to an endothelial cell 'culture shock ' protein of Mr 43
000.
EMBO J. 5, 1465-1472.
McBumey, MW and Ropers, B.J. (1982). Isotation of Male Embryonal
Carcinoma Cells and Their Chromosome Replication Patterns. Dev. Biol. 89, 503--
508.
Mummery, C.L, Feijen, P.T., van der Saag, P.T., van den Brink, C.E. and
de Laat, S.W. (7985} Clonal variants of differentiated P19 embryonal carcinoma
cells exhibit epidermal growth factor receptor kinase activity. Dev. Biol.
109, 402-
410.
Pease, S., Braghetta, P., Gearing, D., Grail, D. and Williams, R. L. (1990).
Isolation of Embryonic Stem {ES) Cells in Media Supplemented with Recombinant
Leukemia Inhibitory Factor (LIF). Dev. Biol. 141, 344-352.
Poirier, F., Chan, C.-T. J., Timmons, P. M., Robertson, E., Evans, M. J. and
Rigby, P. W. J. (1991). The marine H19 gene is activated during embryonic stem
cell differentiation in vitro and at the time of implantation in the
developing embryo.
Development 113, 1105-1114.
Rathjen, P.D., Nichois, J., Toth, S., Edwards, D.R., Heath, J.K. and Smith,
CA 02324591 2000-09-19
WO 99153021 PCTIAU99/00265
112 -
A.G. {1990). Developmentally programmed induction of differentiating
inhibiting
activity and the control of stem cell populations. Genes and Dev. 4, 2308-
2318.
Ringwald, M., Schuh, R., Vestweber, D., Eistetter, H., Lottspeich, F., Engei,
J., Dolz, R., Jahnig, F., Epplen, J., Mayer, S., Muller, C. and Kemler, R.
(1987).
The structure of cell adhesion molecule uvomorulin. Insights into the
molecular.
mechanism of Ca21-dependent cell adhesion. EMBO J. fi, 3647-3653.
Robertson, E., Bradley A., Kuehn, M. and Evans, M. (1986). Germ-fine
transmission of genes introduced into cultured piuripotential cells by
retroviral
vector. Nature 323, 445-448.
Robertson, E.J. (1987). Embryo-derived stem cell fines. in
Teratocarcinomas and Embryonic Stem Cells, A Practical Approach., p71-1 i2 IRL
Press, Oxford.
Rogers, M.B., Hosier, B.A. and Gudas, L.J. (1991 ). Specific expression of a
retinoic acid-regulated, zinc-finger gene, Rex-1, in preimplantation embryos,
trophoblast and spermatocytes. Development 113, 815-824.
Rosen B. and Beddington, R. S. (1993). Whole-mount in situ hybridisation
in the mouse embryo: gene expression in three dimensions. Trends Genet. 9,
162-167.
Rosner, M.H., Vigano, A., Ozato, K, Timmons, RK, Poirer, E, Righy, P. W.J.
and Staudit, L.M. (1990). A POU-domain transciption factor in early stem cells
and
germ cells of the mammalian embryo. Nature 345, 686-692.
Rudnictci, M. A. and McBumey, M. W. (1987}. Cell culture methods and
induction of differentiation of embryonal carcinoma cell lines. In
Teratocarcinomas
and embryonic stem cells: a practical approach. (ed. Robertson, E. J). pp 19-
50.
IRL press, Oxford.
Sassa, S. and Kappas, A. (1977) Induction of aminolevulinate synthase
CA 02324591 2000-09-19
WO 9915302! PCT/AU99/OOZ65
113 -
anmd porphyrins in cultured liver cells maintained in chemically defined
medium.
Permissive effects of hormones on induction process. J. Blot. Chern. 252, 2428-
2436.
Scholer, H. R., Dressier, G. R., Balling, R., Rohdewolild, H. and Gruss, P.
{1990). Oct-4: a germline-specific transcription factor mapping to the mouse
t-complex. EMBO J. 9, 2185-2195.
Sefton, M., Johnson, M.H. and Clayton, L. (1992). Synthesis and
phosphoryiation of uvomorulin during mouse early development. Development
115, 313-318.
Smith, A. G. (1991 ). Culture and Differentiation of Embyonic Stem Cells. J.
Tiss. Cult. Meth. 13, 89-94.
Stewart C. L. (1993). Production of Chimeras between Embryonic Stem
Cells and Embryos. In Guide to Techniques in Mouse Development (eds
Wasserman P. M. and DePamphilis M. L.) Meth. Enzymol. 225, 823-855.
Thomas, P. Q, Johnson, B. V., Rathjen, J. and Rathjen, P. D. (1995).
Sequence, genomic organization, and expression of the novel homeobox gene
Hesxl. J. Biol. Chem. 270, 3869-3875.
Yeom, Y.l., Ha, H-S., Balling, R., Scholer, H. and Artzt, K. (1991 ).
Structure,
expression and chromosomal tocation of the Oct-4 gene. Mech. of Dev. 35,
171-179.
Finally, it is to be understood that various alterations, modifications andlor
additions may be made without departing from the spirit of the present
invention
as outlined herein.
CA 02324591 2000-09-19
WO 99153021 PCTIAU99/00265
I14
TART F I
ES CeIL~ICM EPL P~l~ve
Cells Ectoderm
Oct-4 ~- + + +
lkaline phosplrarase+ + + +
Uvorrtorulin + + + +
.
F8fs ~ - - + +
Rex-1 high + low
lphafetoprotein(AFP- - - -
H19 - - - -
Evxl - - - -
rachyury ' - - - _
SUHSTITZTTE SHEET (Rule 26) (ROIAU)
CA 02324591 2000-09-19
WO 99/53021 PCT/AU99/00265
lI5
TABLE 2
BlastocystsPips oho ~ e~mcra
injectedborn iivcborn
ES; E14TG2a 163 65 40 58
EPL:2 65 33 50 0
EPL;4 ~ 77 50 65 0
EPL;2R 78 22 28 36
EPL; 4R 74 31 42 58
SUBS'ITI'UTE SHEET (Rule 26) (ROIAU)
CA 02324591 2000-09-19
WO 99/53021 PCTIAU99/00265
116
TAHL 3
Matrix composition % EPL cell colonies with
no associated differentiation
Gelatin (0.2%) ~ %
Laminin 49 %
Plasma Fibronectin ~ 51 %
Collagen IV 59 %
Laminin/fibronectin/collagen N 62.5%
mix
SUBSTITUTE SHEET (Rule 26) (ROIAU)
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
117
TART F 4
Maintenance and proliferation of embryonic primitive ectoderm
in response to MEDII
Embryo Embryo Pluripotent
culture conditions Matrix Survival cell maintenance
(%) (%)
DMEM + mLIF Collagen IV 23.5 0
DMEM + 50% MEDII Collagen IV 42 23
SUBSTTTUTE S~'I" (Rule 26) (RO/AU)
CA 02324591 2000-09-19
WO 99153021 PC'T/AU99100265
118
ART F 5
Effect of different treatmentslchromatography of semi-puri~led* HEPG2
conditioned media on
its ability to convert ES cells into X cells
Treatment ES to X cell ~ conversion **
no +
repeated freeze thawing +
acid (pH2.0) +
heat (lhr, 100C) +
DTT (SQmM) +
Ian exchange:
Cation or Anion (various buf~ers.~pH's)
flow through fraction +
eluted fraction -
Reverse phase HPLC
C18 column (0.1%TFA-> 80%acetonitrile)
~
flow through fraction +
~
eluted fraction -
C8 columw(0.1 %TFA -> 80%acetonitrile)
flow through fraction +
eluted fraction -
*The eluate from the 3kD ultrafiltration of HEPG2 conditioned media was
used.
** The ability of the treatedlchromatographed eluate (plus desalted retentate)
to convert ES to X cells was tested using the assay described in the Materials
and Methods.
SLFHSTITUTE SHEET (Rule 26) (ROIALn
CA 02324591 2000-09-19
WO 99153021 PCT/AU99/00265
119
TABLE 6
Amino acid Active sample Control sample
Hydrolysed Unhydrolysed
(pmol) (pmol) (pmol)
Aspartic acid 0 not found 3
Glutamic acid 35.2 8.3 6.2
Serine 2.6 6.1 9.8
Histidine not found not found not found
Glycine 54.4 64.9 not found
Threonine not found not found not found
Aianine 403.3 478.5 I1
Arginine not found not found not found
Tyrosine not found not found not found
Valine 18.1 23.9 30.9
Metr~ionine not found 3.3 I8.3
Pheny3lanine 0 not found 10.4
Isoleucine 29.5 34.6 58.6
Leucine 25.8 27.4 74.3
Lysine not found not found not found
Proline . 478.8 558 25.G
SUBSTITUTE SI~ET (Rule 26) (ROIAU)
CA 02324591 2000-09-19
WO 99/53021 PCTIAU99/00265
120
TA~..E 7
Minimal Range Activity
Active tested
concentration
( wM ) { ~tM )
L-proline (40) 20-1000 +
AMINO ACID
D-Praline 30.75 -
L-Alanine 3g~gg00 -
L-Lysine 55-X00
PROL1NE ANALOG
N-acetly-L-proli ne 64-636 -
trans-4-hydroxy-L-proline 270-550 _
N-t-boc-L-proline 10-1000 _*
L pipechotic 390-15500 -
acid
(PCA)
Sarcosine 10-i 120 -
-
3,4 dehydro-L-proline 1-500 -
pyrrolidine 10-1000 -
PEPTIDE
Pro-ala (250) 20-1000 +
Afa ro
-P (80) 20-1000
Ala-pro-gly ( 40) 40-1000
Pro-OH-pro ( 40-80) 20-1000 +
Pro-gly ( 250') 20-1000 +
Gly-pro ( 40) 20-i 000 +
Giy-pro-ala (40) 20-1000 +
G ly-pro-OH-p (300) 40-5850 +
ro
Gly-pro-arg-pro (80) 40-1000 +
G1y-pro-gly-gly (50) 1-1200 +
Val-ata-pro-gly (40) 40-1000 +
Substance 0.005-500 *
P
(Arg -iiro-iys-pro-gln-gin-phe-phe-gly-leu-met-NHS
SubstanceP free (40) 40-7 000 f
acid
(Arg-pro-1ys-pro-gln-gin-p he-phe-giy-leu-met-OH)
Substance (40) 40-1000 +
P frag.
1-4
(Arg-pro-iys-pro)
Arg-gly-asp 40-4800 -
(RGD)
SUBSTIT'U'TE SHEET (Rule 26) (RO/AU)
CA 02324591 2000-09-19
WU 99153021 PCT/AU99/00265
121
c
..,
~ o
~_L
1 1 1
1 _t _I _t
1.r 1 1 1 1
C
H
a~
s.
to
C
..,
C
o O
O~
O
t 1 r 1 1 f 'E'
O
.
..
wr
1 t 1 1
~1 1 t
C
v U
W
U ~
>
.,
,'~ ~ ~ .,G
O
w w
:o
,e ~ ~ ~ ~~ ~a a
~
~a ~~,p
'~ ~
o ,
~
U > C7 ,~ a. U
a.
~
SUBSTITUTE SF3EET (Rule 26) (RO/AU)
