U.S. patent application number 14/225245 was filed with the patent office on 2014-09-25 for methods of inducing differentiation of stem cells.
This patent application is currently assigned to ES Cell International Pte LTD. The applicant listed for this patent is ES Cell International Pte LTD. Invention is credited to Christine Lindsay Mummery.
Application Number | 20140287495 14/225245 |
Document ID | / |
Family ID | 25646758 |
Filed Date | 2014-09-25 |
United States Patent
Application |
20140287495 |
Kind Code |
A1 |
Mummery; Christine Lindsay |
September 25, 2014 |
Methods of Inducing Differentiation of Stem Cells
Abstract
The present invention relates to methods of inducing
differentiation of stem cells. In particular, the invention relates
to methods of inducing differentiation of embryonic stem cells into
muscle cells or vascular endothelial cells. The invention also
includes cells, cell lines, testing models and culture systems used
in the methods of the present invention and differentiated cells
produced therefrom. The present invention also provides methods of
using the differentiated cells of the present invention for
therapeutic purposes.
Inventors: |
Mummery; Christine Lindsay;
(Bilthoven, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ES Cell International Pte LTD |
Alameda |
CA |
US |
|
|
Assignee: |
ES Cell International Pte
LTD
Alameda
CA
|
Family ID: |
25646758 |
Appl. No.: |
14/225245 |
Filed: |
March 25, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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PCT/AU02/00978 |
Jul 23, 2002 |
|
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14225245 |
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Current U.S.
Class: |
435/347 ;
435/366 |
Current CPC
Class: |
C12N 5/069 20130101;
C12N 5/0606 20130101; C12N 2501/10 20130101; C12N 5/0658 20130101;
C12N 2501/155 20130101; C07K 14/435 20130101; C12N 2502/28
20130101; C12N 5/0603 20130101; C12N 2510/00 20130101; A61P 9/00
20180101; C12N 5/0657 20130101; A61P 9/10 20180101; C12N 2506/02
20130101; A61P 43/00 20180101; A61K 35/12 20130101; C12N 2502/1329
20130101; C12N 2502/02 20130101; A61P 3/10 20180101; A61P 9/04
20180101 |
Class at
Publication: |
435/347 ;
435/366 |
International
Class: |
C12N 5/0735 20060101
C12N005/0735; C12N 5/073 20060101 C12N005/073 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 24, 2001 |
AU |
PR6560 |
Mar 18, 2002 |
AU |
PS1180 |
Claims
1-44. (canceled)
45. A co-culture comprising human embryonic stem cells and END2
cells.
46. The co-culture of claim 45, further comprising
cardiomyocytes.
47. The co-culture of claim 45, further comprising cells expressing
ANF, .alpha.1c, Kv4.3 and KvLQT1.
48. The co-culture of claim 46, wherein the cardiomyocytes are
beating.
49. The co-culture of claim 45 further comprising endothelial
cells.
50. The co-culture of claim 45, wherein the END2 cells have been
treated with mitomycin C before being co-cultured with the human
embryonic stem cells.
51. The co-culture of claim 45 further comprising FGF or BMP.
52. A cell culture comprising human embryonic stem cells and media
conditioned by END2 cells.
53. The co-culture of claim 52, further comprising
cardiomyocytes.
54. The co-culture of claim 52, further comprising cells expressing
ANF, .alpha.1c, Kv4.3 and KvLQT1.
55. The co-culture of claim 53, wherein the cardiomyocytes are
beating.
56. The co-culture of claim 52 further comprising endothelial
cells.
Description
[0001] The present invention relates to methods of inducing
differentiation of stem cells. In particular, the invention relates
to methods of inducing differentiation of embryonic stem cells into
muscle cells or vascular endothelial cells. The invention also
includes cells, cell lines, testing models and culture systems used
in the methods of the present invention and differentiated cells
produced therefrom. The present invention also provides methods of
using the differentiated cells of the present invention for
therapeutic purposes.
INTRODUCTION
[0002] Stem cells are undifferentiated cells which can give rise to
a succession of mature functional cells. Embryonic stem (ES) cells
are derived from the embryo and are pluripotent, thus possessing
the capability of developing into any organ, cell type or tissue
type. The process of differentiation in stem cells involves
selective development of immature cells to committed and fully
mature cells of various lineages. Derivatives of such lineages
include, muscle, neural, skeletal, blood (hematopoietic),
endothelial and epithelial cells. Differentiation of stem cells is
known be triggered by various growth factors and regulatory
molecules.
[0003] During differentiation the expression of stem cell specific
genes and markers are often lost and cells acquire gene expression
profiles of somatic cells or their precursors. In some cases,
"master" genes have been described which control differentiation
versus self-renewal.
[0004] Whilst differentiation of some lineage specific stem cells
can be induced with a degree of certainty, a differentiation
outcome of a population of pluripotent stem cells is less
predictable. Placing the cells under conditions which induce
specific cell types has been one form of an attempt to regulate the
differentiation outcome. These conditions include growing the cells
to high or low density, changing media, introducing or removing
cytokines, hormones and growth factors, creating an environment
which suits differentiation toward a specific cell type, such as
providing a suitable substrate.
[0005] Generally, when a stem cell culture is induced to
differentiate, the differentiated population is analysed for
particular cell types by expression of genes, markers or phenotypic
analysis. In any case, the respective cell types may then be
selectively cultured to enrich their percentage population to
eventually obtain a single cell type and culture.
[0006] The induction of a specific differentiated cell type can be
useful for transplantation or drug screening and drug discovery in
vitro. Methods of inducing differentiation in stem cells and muscle
cells produced therefrom may be used for the study of cellular and
molecular biology of tissue development, for the discovery of genes
and proteins such as differentiation factors that play a role in
tissue development and regeneration.
[0007] In particular, the induction of stem cells to differentiate
into muscle cells (myocytes) is useful for muscle transplantation
and therapeutic purposes, as well as providing potential human
disease models in culture (e.g. for testing pharmaceuticals). The
induction of cardiomyocyte differentiation in stem cells is
especially useful in developing therapeutic methods and products
for heart disease and abnormal heart conditions. However, the
molecular pathways that lead to specification and terminal
differentiation of specific cell types, such as myocytes, from
embryonic stem cells during development are not entirely clear.
[0008] Therefore there remains a need for providing effective
methods of inducing differentiation of stem cells into specific
cell types, such as myocytes or endothelial cells.
SUMMARY OF THE INVENTION
[0009] In one aspect of the present invention there is provided a
method of inducing differentiation of a stem cell, the method
including: [0010] culturing a stem cell in the presence of an
embryonic cell and/or extracellular medium of an embryonic cell,
under conditions that induce differentiation of the stem cell.
[0011] Preferably, the stem cell is an embryonic, human stem cell.
More preferably, the stem cell is induced to differentiate into a
myocyte (muscle cell), vascular endothelial cell or a
haematopoietic cell. In the methods of the present invention as
hereinbefore described, the embryonic cell is preferably derived
from embryonic endoderm or ectoderm. More preferably, the embryonic
cell is derived from extraembryonic tissue.
[0012] In the methods of the present invention the stem cell is
preferably grown in the presence of an embryonic, endoderm cell
and/or extracellular conditioned medium of an embryonic, endoderm
cell to induce differentiation of the stem cell into a
cardiomyocyte or a haematopoietic cell. More preferably, the stem
cell is co-cultured in the presence of the embryonic cell. In the
methods of the present invention the stem cell is preferably plated
on a confluent monolayer of embryonic cells and allowed to grow in
culture to induce differentiation of the stem cell.
[0013] Alternatively, the stem cell is grown in the presence of an
embryonic, ectoderm cell and/or extracellular medium of an
embryonic, ectoderm cell to induce differentiation of the stem cell
into a skeletal muscle cell.
[0014] In yet another preferred embodiment, the stem cell is grown
in the presence of an embryonic, ectoderm and/or endoderm cell,
and/or extracellular medium of an embryonic, ectoderm and/or
endoderm cell to induce differentiation of the stem cell into a
vascular endothelial cell.
[0015] In a preferred aspect of the present invention there is
provided a method of inducing muscle cell differentiation of a stem
cell, the method including: [0016] culturing a stem cell in the
presence of an embryonic cell and/or extracellular medium of an
embryonic cell, under conditions that induce differentiation of the
stem cell into a muscle cell.
[0017] Preferably, the stem cell is grown in the presence of an
embryonic, endoderm cell and/or extracellular medium of an
embryonic, endoderm cell to induce differentiation of the stem cell
into a cardiomyocyte (cardiac muscle cell). More, preferably the
embryonic cell is extraembryonic. The embryonic cell is preferably
derived from visceral endoderm, is a cell with visceral endoderm
like properties or is derived from an embryonic cell line with
characteristics of visceral endoderm. More preferably, the stem
cell is co-cultured in the presence of the embryonic cell.
[0018] Alternatively, the stem cell is grown in the presence of an
embryonic, ectoderm cell and/or extracellular medium of an
embryonic, ectoderm cell to induce differentiation of the stem cell
into a skeletal muscle cell. More preferably, the embryonic cell is
derived from extraembryonic tissue.
[0019] Another aspect of the present invention is an embryonic cell
for use in the methods of the present invention. Preferably, the
embryonic cell is derived from embryonic or extraembryonic endoderm
or ectoderm. Preferably, the embryonic cell is derived from
visceral endoderm or is a cell with visceral endoderm like
properties. More preferably, the embryonic cell is derived from a
cell line with characteristics of visceral endoderm, such as the
END-2 cell line (Mummery et al, 1985, Dev Biol. 109:402-410).
[0020] In a further preferred aspect of the present invention there
is provided a method of inducing differentiation of a stem cell,
the method including: [0021] culturing a stem cell in the presence
of a factor derived from an embryonic cell or extracellular medium
of an embryonic cell under conditions that induce differentiation
of the stem cell.
[0022] The present invention further provides an isolated factor
that is secreted from an embryonic cell or is isolated from
extracellular medium according to the present invention for use in
a method of inducing differentiation of a stem cell.
[0023] In yet another aspect of the invention, there is provided a
differentiated cell produced according to the methods as
hereinbefore described. Preferably, the differentiated cell is a
cardiomyocyte, skeletal muscle cell, vascular endothelial cell or a
haematopoietic cell. The present invention also provides
differentiated cells produced according to the methods of the
invention that may be used for transplantation, cell therapy or
gene therapy. Preferably, the invention provides a differentiated
cell produced according to the methods of the invention that may be
used for therapeutic purposes, such as in methods of restoring
cardiac function in a subject suffering from a heart disease or
condition.
[0024] Another aspect of the invention is a method of treating or
preventing a cardiac disease or condition, the method including
[0025] introducing an isolated cardiomyocyte cell and/or a cell
capable of differentiating into a cardiomyocyte cell into cardiac
tissue of a subject.
[0026] Preferably, the cardiomyocyte is produced by the
differentiation of a stem cell according to methods as hereinbefore
described. It is preferred that the subject is suffering from a
cardiac disease or condition. In the method of the present
invention, the isolated cardiomyocyte cell is preferably
transplanted into damaged cardiac tissue of a subject. More
preferably, the method results in the restoration of cardiac
function in a subject.
[0027] In yet another preferred aspect of the invention there is
provided a method of repairing cardiac tissue, the method including
[0028] introducing an isolated cardiomyocyte cell and for a cell
capable of differentiating into a cardiomyocyte cell into damaged
cardiac tissue of a subject.
[0029] Preferably, the cardiomyocyte is produced by the
differentiation of a stem cell according to methods as hereinbefore
described. It is preferred that the subject is suffering from a
cardiac disease or condition. In the method of the present
invention, the isolated cardiomyocyte cell is preferably
transplanted into damaged cardiac tissue of a subject. More
preferably, the method results in the restoration of cardiac
function in a subject.
[0030] The invention also provides methods of treating vascular
diseases and muscular diseases by transplanting differentiated to
vascular endothelial cells or to skeletal muscle cells or
progenitors of these cells.
[0031] The present invention preferably also provides a myocardial
model for testing the ability of stem cells that have
differentiated into cardiomyocytes to restore cardiac function.
[0032] The present invention further provides a cell composition
including a differentiated cell produced by the method of the
present invention, and a carrier.
FIGURES
[0033] FIG. 1A shows a phase contrast micrograph of human embryonic
stem (hES) in co-culture with END-2 cells after a period of 13
days. The differentiated stem cells have mixed morphology but with
a relatively high proportion of epithelial-like cells. The
epithelial cells swell to fluid-filled cysts and between these
cells are patches of cardiomyocytes. Cross section of the colony
shown is about 2 mm (40.times. objective). Scale bar=100 .mu.m.
[0034] FIG. 1B shows a phase contrast micrograph of human embryonic
stem (hES) in co-culture with END-2 cells after a period of two to
three weeks. Increasing patches of beating cardiac muscle cells
(cardiomyocytes; bm) are present. The beating rate observed was
approximately 60 beats per minute. (20.times. objective). Scale
bar=100 .mu.m.
[0035] FIG. 2 shows cells stained positively with .alpha.-actinin,
confirming that they are indeed muscle cells. Scale bar=100
.mu.m.
[0036] FIG. 3 shows co-cultures of stem cells with the mouse
visceral endoderm-like cell line END-2. (a) P19 EC in normal
monolayer culture, 3 days after initiation of co-culture with END-2
cells and after 10 days, when beating muscle (B.M.) is evident. (b)
mES cell lineR1 in monolayer on its normal `feeder` cells (SNL), 3
days after initiation of co-culture and 2 days later, when beating
muscle is evident. (c) as (b), with the exception that B.M. is
evident on day 7 after aggregation. (d) GCT27X human EC cell line
on mouse embryonic fibroblast (MEF) feeder cells, 3 days after
initiation of co-culture and after 16 days. No beating muscle is
present. (e) hES cells on MEF feeders, 3 days after initiation of
END-2 co-culture and beating muscle formed after 11 days.
[0037] FIG. 4 shows electrophysiological characteristics of
cardiomyocytes from stem cells. Repetitive action potentials
recorded from spontaneously beating areas. (a) In mouse P19 EC
cell-derived cardiomyocytes. (b) In an aggregate of hES-derived
cardiomyocytes. (c) Phase contrast image of the beating area in the
hES culture from which the recording showed in (b) was derived.
(Note the height of the protruding structure where the beating
region is located, 20.times. objective.)
[0038] FIG. 5 shows isolated cardiomyocytes: (a) exhibiting sharp
edges and well-defined sarcomeres in contrast with cells cultured
for 2 days (b) which had disorganized sarcomeric patterning. (a) is
a phase contrast image of multiple cells after isolation and
fixation. (b) represents a single cell, digitally magnified
2.times. compared with (a).
[0039] FIG. 6 shows Immunocytochemistry on adult human primary
atrial cardiomyocytes and stem cell-derived cardiomyocytes. Primary
atrial cardiomyocytes stained positive for sarcomeric proteins
including (green) .alpha.-actinin, (red) mlc-2a (a) and tropomyosin
(b). Cell DNA was stained with (blue) Hoechst to distinguish normal
and apoptotic cells. Cells cultured for 2 days had a disorganized
tropomyosin sarcomeric patterning and diffuse antibody staining
(c). mES-derived cardiomyocytes also show sharp banding when
stained with .alpha.-actinin (d) but in hES-derived cardiomyocytes
.alpha.-actinin is diffuse and poorly banded (not shown). (e) shows
overall extensive .alpha.-actinin staining in hES-derived
cardiomyocytes at low magnification.
[0040] FIG. 7 shows haemodynamic assessment of left ventricular
function in mice. (a) Normal loop representing the relationship
between volume and pressure changes in the mouse heart: indicated
are the valvular events and stages during one cycle of contraction
and relaxation. (b) Pressure volume relationship 4 weeks
post-myocardial infarction: note the difference in the shape of the
loop and the alterations in both contraction and relaxation.
[0041] FIG. 8 shows expression of cardiomyocyte marker mRNA in
co-cultures of hES and END-2 cells by RT-PCR.
[0042] FIG. 9 shows analysis of A. action potentials and ion
channels by patch clamp electrophysiology and B. real-time analysis
of Ca.sup.2+.
[0043] FIG. 10 shows effects of END-2 conditioned medium on P19
embryonal carcinoma cells. A. Induction of beating muscle in P19EC
aggregates. Results from two independent clones (P19 EC and P19
clone 6 EC) on day 7 and 8 as indicated, are shown. All aggregates
beat in the presence of DMSO on day 10. B. Northern blot showing
induction of Brachyury T by END-2 conditioned medium in P19EC
aggregates. DMSO also induces cardiomyocyte differentiation and is
shown as a control. Both END-2 CM and DMSO induced Brachyury T
expression are blocked by activin.
DETAILED DESCRIPTION OF THE INVENTION
[0044] In one aspect of the present invention there is provided a
method of inducing differentiation of a stem cell, the method
including: [0045] culturing a stem cell in the presence of an
embryonic cell and/or extracellular medium of an embryonic cell,
under conditions that induce differentiation of the stem cell.
[0046] Stem cells usually require co-culture with a fibroblast
feeder layer to maintain their undifferentiated state. Those feeder
layers that do not adequately maintain this state result in stem
cells loosing their undifferentiated characteristics to non-obvious
phenotypes. Applicants have found that culturing stem cells with
embryonic cells can provide a determining factor to the outcome of
differentiated cells in culture. This control has never been seen
with fibroblast cells.
[0047] The term "inducing differentiation of a stem cell" as used
herein is taken to mean causing a stem cell to develop into a
specific differentiated cell type as a result of a direct or
intentional influence on the stem cell. Influencing factors that
may induce differentiation in a stem cell can include cellular
parameters such as ion influx, a pH change and/or extracellular
factors, such as secreted proteins, such as but not limited to
growth factors and cytokines that regulate and trigger
differentiation. It may include culturing the cell to confluence
and may be influenced by cell density.
[0048] In the methods of the present invention a stem cell is
undifferentiated prior to culturing and is any cell capable of
undergoing differentiation. The stem cell may be selected from the
group including, but not limited to, embryonic stem cells,
pluripotent stem dells, haematopoietic stem cells, totipotent stem
cells, mesenchymal stem cells, neural stem cells, or adult stem
cells.
[0049] The stem cell is preferably a human embryonic stem cell
which may be derived directly from an embryo or from a culture of
embryonic stem cells. For example, the stem cell may be derived
from a cell culture, such as human embryonic stem cells (hES) cells
(Reubinoff et al., Nature Biotech. 16:399-404 2000). Whilst, the
stem cell may be derived from other animals, they are most
preferably human embryonic stem cells. The stem cell may be derived
from an embryonic cell line or embryonic tissue. The embryonic stem
cells may be cells which have been cultured and maintained in an
undifferentiated state. Such cells have been described in
PCT/AU99/00990, PCT/AU00/01510, PCT/AU01/00735 and PCT/AU01/00278,
the contents of which are incorporated herein by reference.
[0050] The stem cells suitable for use in the present methods may
be derived from a patient's own tissue. This would enhance
compatibility of differentiated tissue grafts derived from the stem
cells with the patient. The stem cells may be genetically modified
prior to use through introduction of genes that may control their
state of differentiation prior to, during or after their exposure
to the embryonic cell or extracellular medium from an embryonic
cell. They may be genetically modified through introduction of
vectors expressing a selectable marker under the control of a stem
cell specific promoter such as Oct-4. The stem cells may be
genetically modified at any stage with markers so that the markers
are carried through to any stage of cultivation. The markers may be
used to purify the differentiated or undifferentiated stem cell
populations at any stage of cultivation.
[0051] The stem cell can be induced to differentiate into a cell
type selected from the group including muscle cells, endothelial
cells, such as vascular endothelial cells, epithelial cells, blood
cells (haematopoietic cells) or neural cells. Preferably, the stem
cell is induced to differentiate into a myocyte (muscle cell) or a
vascular endothelial cell. More preferably, the stem cell is
induced to differentiate into a cardiomyocyte or a skeletal muscle
cell.
[0052] In a preferred embodiment of the present invention there is
provided a method of inducing differentiation of a stem cell, the
method including: [0053] culturing a stem cell in the presence of
an embryonic cell under conditions that induce differentiation of
the stem cell.
[0054] The embryonic cell used in the present method includes an
embryonic cell derived from an embryo or a cell derived from
extraembryonic tissue. The term "embryo" is defined as any stage
after fertilisation up to 2 weeks post conception in mammals. It
develops from repeated division of cells and includes the stages of
a blastocyst stage which comprises an outer trophectoderm and an
inner cell mass (ICM). The embryo may be an in vitro fertilised
embryo or it may be an embryo derived by transfer of a somatic cell
or cell nucleus into an enucleated oocyte preferably of human or
non-human origin. Extraembryonic tissue includes cells produced by
the embryo that make up the placenta.
[0055] In a preferred embodiment of the invention, the embryonic
cell is derived from embryonic, preferably extraembryonic, endoderm
or ectoderm. More preferably, the embryonic cell is derived from
visceral endoderm tissue or visceral endoderm like tissue isolated
from an embryo. Preferably visceral endoderm may be isolated from
early postgastrulation embryos, such as mouse embryo (E7.5).
Visceral endoderm or visceral endoderm like tissue can be isolated
as described in Roelen et al, 1994 Dev. Biol. 166:716-728.
Characteristically the visceral endoderm may be identified by
expression of alphafetoprotein and cytokeratin ENDO-A). The
embryonic cell may be an embryonal carcinoma cell, preferably one
that has visceral endoderm properties.
[0056] The embryonic cell may be derived from a cell line or cells
in culture. The embryonic cell may be derived from an embryonic
cell line, preferably a cell line with characteristics of visceral
endoderm, such as the END-2 cell line (Mummery et al, 1985, Dev
Biol. 109:402-410). The END-2 cell line was established by cloning
from a culture of P19 EC cells treated as aggregates in suspension
(embryoid bodies) with retinoic acid then replated (Mummery et al,
1985, Dev Biol. 109:402-410). The END-2 cell line has
characteristics of visceral endoderm (VE), expressing
alpha-fetoprotein (AFP) and the cytoskeletal protein ENDO-A.
Accordingly it is most preferred that the embryonic cell is derived
from the END-2 cell line. These cell have been found to be
particularly useful for inducing differentiation of a human stem
cell to a cardiomyocyte (cardiac muscle cell).
[0057] The embryonic cell may be an ectoderm cell, which can be
isolated according to methods described in Roelen et al 1994, Dev.
Biol. 166:716-728. Ectoderm cells are known to express oct-4 and
have alkaline phosphatase activity and they also have SSEA-1 on
their cell surface. Therefore, ectoderm cells can be identified and
isolated based on the above characteristics. Ectoderm cells may
secrete (growth) factors that induce differentiation to skeletal
muscle or vascular endothelial cells. It is preferred that the
ectoderm cells are derived from E7.5, embryonic mouse tissue.
[0058] Accordingly, in another aspect of the present invention
there is provided an embryonic cell for use in the methods of the
present invention. Preferably, the embryonic cell is derived from
embryonic endoderm or ectoderm as discussed above.
[0059] In the present invention and methods as hereinbefore
described, the stem cell and embryonic cell are cultured to induce
differentiation into a specific cell type. Preferably, the stem
cell and embryonic cell are co-cultured in vitro. This typically
involves introducing the stem cell to an embryonic cell monolayer
produced by proliferation of the embryonic cell in culture.
Preferably, the embryonic cell monolayer is grown to substantial
confluence and the stem cell is allowed to grow in the presence of
extracellular medium of the embryonic cells for a period of time
sufficient to induce differentiation of the stem cell to a specific
cell type. Alternatively, the stem cell may be allowed to grow in
culture containing the extracellular medium of the embryonic
cell(s), but not in the presence of the embryonic cell(s). The
embryonic cells and stem cells may be separated from each other by
a filter or an acellular matrix such as agar.
[0060] In the methods of the present invention the stem cell is
preferably plated on a monolayer of embryonic cells and allowed to
grow in culture to induce differentiation of the stem cell. More
preferably, the monolayer is confluent and is mitogenically
inactive.
[0061] Conditions for obtaining differentiated embryonic stem cells
are those which are non-permissive for stem cell renewal, but do
not kill stem cells or drive them to differentiate exclusively into
extraembryonic lineages. A gradual withdrawal from optimal
conditions for stem cell growth favours differentiation of the stem
cell to specific cell types. Suitable culture conditions may
include the addition of DMSO, retinoic acid, FGFs or BMPs in
co-culture which could increase differentiation rate and/or
efficiency.
[0062] The cell density of the embryonic cell layer affects its
stability and performance. The embryonic cells should preferably be
confluent. More preferably, the embryonic cells are grown to
confluence and are then exposed to an agent which prevents further
division of the cells, such as mitomycin C. The embryonic monolayer
layer is preferably established 2 days prior to addition of the
stem cell(s). The stem cells are preferably dispersed and then
introduced to a monolayer of embryonic cells. More preferably, the
stem cells and embryonic cells are co-cultured for a period of two
to three weeks until a substantial portion of the stem cells have
differentiated. Preferably, the stem cell is induced to
differentiate into a myocyte (muscle cell) including cardiomyocytes
and skeletal muscle cells, a vascular endothelial cell or a
haematopoietic cell. It is preferred that the embryonic cell is
derived from extraembryonic tissue and more preferably from
embryonic endoderm or ectoderm.
[0063] In another preferred embodiment of the present invention
there is provided a method of inducing differentiation of a stem
cell, the method including: [0064] culturing a stem cell in the
presence of extracellular medium of an embryonic cell under
conditions that induce differentiation of the stem cell.
[0065] The term "extracellular medium of an embryonic cell" as used
herein is taken to mean conditioned medium produced from growing an
embryonic cell as herein described in a medium for a period of time
so that extracellular factors, such as secreted proteins, produced
by the embryonic cell are present in the conditioned medium. The
medium can include components that encourage the growth of the
cells, for example basal medium such as Dulbecco's minimum
essential medium, Ham's F12, or foetal calf serum.
[0066] In an even further preferred aspect of the present invention
there is provided a method of inducing differentiation of a stem
cell, the method including: [0067] culturing a stem cell in the
presence of a factor derived from an embryonic cell or
extracellular medium of an embryonic cell under conditions that
induce differentiation of the stem cell.
[0068] The extracellular medium preferably includes cellular
factors, such as secreted proteins, that are capable of inducing
differentiation of a stem cell. Such secreted proteins will
typically bind receptors on a cell surface to trigger intracellular
pathways which can initiate differentiation of the cell. Examples
of suitable extracellular factors include lhh and BMP2 as described
in Dyer et al 2001, Dev. 128: 1717-1730.
[0069] In another aspect of the present there is provided an
isolated factor that is secreted from an embryonic cell or isolated
from extracellular medium according to the present invention for
use in a method of inducing differentiation of a stem cell.
Suitable isolated factors may be selected by their ability to
induce differentiation of a stem cell. For example, culture cell
assay systems can be used to identify protein fractions and
specific factors that are capable of inducing differentiation of a
stem cell. The factors may include secreted proteins that are
present in the extracellular medium of an embryonic cell. Suitable
proteins may be extracted and purified by conventional methods
known to those skilled in the field.
[0070] In another preferred aspect of the present invention there
is provided a method of inducing muscle cell differentiation of a
stem cell, the method including: [0071] culturing a stem cell in
the presence of an embryonic cell and/or extracellular medium of an
embryonic cell, under conditions that induce differentiation of the
stem cell into a muscle cell.
[0072] In a preferred, embodiment the stem cell is induced to
differentiate in to a cardiomyocyte cell. The applicants have found
that culturing the stem cell with embryonic, preferably
extraembryonic, endoderm cells causes a preferential induction of
differentiation toward specific cell types, in particular toward
muscle cells. It is most preferred that this combination of stem
cell and embryonic endoderm cells induces differentiation toward
cardiomyocytes. It is preferred that the stem cell is human,
preferably a human embryonic stem cell (hES). More preferably, the
stem cell is co-cultured with the embryonic cell. This is typically
achieved by introducing dispersed stem cells to a culture medium
with a monolayer of suitable embryonic cells. More preferably, the
monolayer is of confluent embryonic cells.
[0073] In an even further preferred embodiment the embryonic cell
is an endoderm cell derived from visceral endoderm or is an
embryonic cell with visceral endoderm properties. More preferably,
the visceral endoderm cells are derived from E7.5 mouse embryo. The
embryonic cell may be an embryonal carcinoma cell, preferably one
that has visceral endoderm properties. More preferably, the
embryonic cell is derived from a cell line or cells in culture. The
embryonic cell may be derived from an embryonic cell line,
preferably a cell line with characteristics of visceral endoderm,
such as the END-2 cell line (Mummery et al, 1985, Dev Biol.
109:402-410). More, preferably the embryonic cell is derived from
extraembryonic tissue and more preferably is derived from visceral
endoderm. Endoderm cells are typically adjacent to sites of heart
formation in vertebrates. In individuals where endoderm
differentiation is defective or absent, the heart develops
abnormally.
[0074] In order to induce differentiation of the stem cell to a
cardiomyocyte it is preferable to introduce the stem cell to an
extraembryonic, endoderm cell monolayer in culture. The monolayer
is produced by proliferation of the embryonic cell derived from
embryonic endoderm, more preferably extraembryonic endoderm. The
embryonic endoderm is preferably extraembryonic, visceral endoderm.
More preferably, the cell monolayer is produced by END-2 cells. It
is preferred that the embryonic cells are cultured and passaged
before allowing them to grow to form a monolayer. The monolayer is
preferably grown to confluence in a suitable medium, such as DMEM
or M16 medium. The monolayer may then be treated with certain
agents to prevent further division of the cells. For instance, the
monolayer can be treated with mitomycin and then the stem cell can
be plated on the mitogenically inactive monolayers.
[0075] The stem cells are allowed to grow in the presence of
extracellular medium of the endoderm cells for a period of time
sufficient to induce differentiation of the stem cell to a
cardiomyocyte, that is most preferably beating. Most preferably,
the co-culturing is carried out for about two to three weeks and
the medium is preferably replaced regularly such as every 5 to 6
days with fresh medium.
[0076] Alternatively, the stem cell may be allowed to grow in
culture containing the extracellular medium of the endoderm cells,
but not in the presence of the endoderm cells. Therefore, the stem
cell may be grown in the presence of extracellular medium of an
embryonic, endoderm cell to induce differentiation of the stem cell
into a muscle cell such as a cardiomyocyte (cardiac muscle
cell).
[0077] In the methods of the present invention the cardiomyocyte
cells produced are preferably beating. Cardiomyocytes are a
differentiated cell type derived from stem cells. Muscle cells,
including cardiomyocytes, can be fixed and stained with
.alpha.-actinin antibodies to confirm muscle phenotype.
.alpha.-troponin, .alpha.-tropomysin and .alpha.-MHC antibodies
also give characteristic muscle staining. Preferably, the
cardiomyocytes are fixed according to methods known to those
skilled in the art. More preferably, the cardiomyocytes are fixed
with paraformaldehyde, preferably with about 2% to about 4%
paraformaldehyde. Ion channel characteristics and action potentials
of muscle cells can be determined by patch clamp, electrophysiology
and RT-PCR.
[0078] Stem cells from which cardiomyocytes are to be derived can
be genetically modified to bear mutations in, for example, ion
channels (this causes sudden death in humans). Cardiomyocytes
derived from these modified stem cells will thus be abnormal and
yield a culture model for cardiac ailments associated with
defective ion channels. This would be useful for basic research and
for testing pharmaceuticals. Likewise, models in culture for other
genetically based cardiac diseases could be created. Cardiomyocytes
produced in the present methods can also be used for
transplantation and restoration of heart function.
[0079] For instance, Ischaemic heart disease is the leading cause
of morbidity and mortality in the western world. Cardiac ischaemia
caused by oxygen deprivation and subsequent oxygen reperfusion
initiates irreversible cell damage, eventually leading to
widespread cell death and loss of function. Strategies to
regenerate damaged cardiac tissue by cardiomyocyte transplantation
may prevent or limit post-infarction cardiac failure. The methods
of inducing stem cells to differentiate into cardiomyocytes, as
hereinbefore described would be useful for treating such heart
diseases. Cardiomyocytes produced by the present methods may also
be used in a myocardial infarction model for testing the ability to
restore cardiac function.
[0080] The present invention preferably provides a myocardial model
for testing the ability of stems cells that have differentiated
into cardiomyocytes to restore cardiac function. In order to test
the effectiveness of cardiomyocyte transplantation in vivo, it is
important to have a reproducible animal model with a measurable
parameter of cardiac function. The parameters used should clearly
distinguish control and experimental animals (see for example
Palmen et al. (2001), Cardiovasc. Res. 50, 516-524) so that the
effects of transplantation can be adequately determined. PV
relationships are a measure of the pumping capacity of the heart
and may be used as a read-out of altered cardiac function following
transplantation.
[0081] A host animal, such as but not limited to, an
immunodeficient mouse may be used as a `universal acceptor` of
cardiomyocytes from various sources.
[0082] Preferably, the cardiomyocytes are produced by the method of
the present invention.
[0083] The myocardial model of the present invention is preferably
designed to assess the extent of cardiac repair following
transplant of cardiomyocytes or suitable progenitors into a
suitable host animal. More preferably, the host animal is an
immunodeficient animal created as a model of cardiac muscle
degeneration following infarct that is used as a universal acceptor
of the differentiated cardiomyocytes. This animal can be any
species including but not limited to murine, ovine, bovine, canine,
porcine and any non-human primates. Parameters used to measure
cardiac repair in these animals may include, but are not limited
to, electrophysiological characteristic of heart tissue or various
heart function. For instance, contractile function may be assessed
in terms of volume and pressure changes in a heart. Preferably,
ventricular contractile function is assessed. Methods of assessing
heart function and cardiac tissue characteristics would involve
techniques also known to those skilled in the field.
[0084] In another aspect of the invention there is provided a
method of treating or preventing a cardiac disease or condition in
a patient, the method including [0085] introducing a cardiomyocyte
cell and/or a cardiomyocyte progenitor or a cell capable of
differentiating into a cardiomyocyte into cardiac tissue of a
patient.
[0086] The term "treating or preventing" as used herein means
alleviating or reducing the symptoms of the condition that is being
treated or prevented.
[0087] Preferably, the cardiomyocyte is produced by the
differentiation of a stem cell according to methods as hereinbefore
described. It is preferred that the subject is suffering from a
cardiac disease or condition. In the method of the present
invention, the isolated cardiomyocyte cell is preferably
transplanted into damaged cardiac tissue of a subject. More
preferably, the method results in the restoration of cardiac
function in a subject.
[0088] A "cell that is capable of differentiating into a
cardiomyocyte" or a cardiomyoctye progenitor may include a stem
cell that has been co-cultured with visceral endoderm and/or
extracellular medium of visceral endoderm but has not completed
differentiation toward the cardiomyocyte.
[0089] Suitable cardiac diseases may include, but are not limited
to cardiac infarction or cardiac hypertrophy.
[0090] In yet another preferred aspect of the invention there is
provided a method of repairing damaged cardiac tissue, the method
including [0091] introducing a cardiomyocyte cell and/or a
cardiomyocyte progenitor or a cell capable of differentiating into
a cardiomyocyte into the damaged cardiac tissue of a subject.
[0092] Preferably, the cardiomyocyte is produced by the
differentiation of a stem cell according to methods as hereinbefore
described. It is preferred that the subject is suffering from a
cardiac disease or condition. In the method of the present
invention, the isolated cardiomyocyte cell is preferably
transplanted into damaged cardiac tissue of a subject. More
preferably, the method results in the restoration of cardiac
function in a subject.
Damaged Skeletal Muscle Tissue May Result from Muscular
Dystrophy.
[0093] In yet another preferred aspect there is provided a method
of inducing differentiation of a stem cell to a skeletal muscle
cell, the method including: [0094] culturing a stem cell in the
presence of an embryonic ectoderm cell and/or extracellular medium
of an embryonic ectoderm cell, under conditions that induce
differentiation of the stem cell into a skeletal muscle cell.
[0095] Ectoderm cells can be isolated according to methods
described in Roelen et al 1994, Dev. Biol. 166:716-728. Ectoderm
cells are known to express oct-4 and have alkaline phosphatase
activity and they also have SSEA-1 on their cell surface.
Therefore, ectoderm cells can be identified and isolated based on
the above characteristics. Ectoderm cells may secrete (growth)
factors that induce differentiation to skeletal muscle. It is
preferred that the ectoderm cells are derived from E7.5, embryonic
mouse tissue. More preferably, the ectoderm cells are co-cultured
with the stem cells using similar methods as discussed earlier. An
ectoderm monolayer is preferably established in culture and
preferably dispersed stem cells are introduced to the culture for a
period of time sufficient to induce differentiation of the stem
cells to skeletal muscle cells.
[0096] Skeletal muscle cells are typically elongated, multinucleate
cells that "twitch" and express MF20. When skeletal muscle cells
are stained with .alpha.-actinin they normally produce a striped
staining pattern. Skeletal muscle cells produced by the methods of
the present invention can be used for transplantation in the
treatment of muscle disease.
[0097] In another aspect of the invention there is provided a
method of a method of treating or preventing muscle disease in a
patient, said method comprising: [0098] introducing to the muscle
of the patient, a skeletal muscle cell and/or a skeletal muscle
cell progenitor that has been co-cultured in the presence of
embryonic ectoderm cells and/or extracellular medium of embryonic
ectoderm cells.
[0099] Preferably, the skeletal muscle cells have been
differentiated from a stem cell co-cultured under the conditions
described above.
[0100] Muscle disease may be due to muscular dystrophy. However,
the method of treatment may be applicable to any condition that
requires regeneration and renewal of muscle cells.
[0101] In yet another preferred aspect there is provided a method
of inducing differentiation of a stem cell to a vascular
endothelial cell, the method including: [0102] culturing a stem
cell in the presence of an embryonic ectoderm and/or endoderm cell,
and/or extracellular medium of an embryonic, ectoderm and/or
endoderm cell to induce differentiation of the stem cell into a
vascular endothelial cell.
[0103] More, preferably the embryonic cell is derived from
extraembryonic ectoderm and/or endoderm tissue. Preferably,
embryonic tissue is derived form embryonic E7.5 mouse. The
embryonic ectoderm or endoderm cell may be obtained as previously
discussed. The first vascular network in the embryo forms adjacent
to visceral extraembryonic endoderm in the yolk sac, which produce
factors affecting endothelial cells like TGF.beta. and VEGF.
Therefore, more preferably the embryonic cell used in the above
method is derived from visceral extraembryonic endoderm or is a
cell with visceral cell like properties. The embryonic endoderm
and/or ectoderm cell is preferably co-cultured with the stem cell
using methods previously discussed. However, VEGF may be added to
the culture medium to promote vascular endothelial cell growth in
culture.
[0104] Vascular endothelial cells produced by the method of the
invention can be identified by being capable of forming vascular
networks sometimes containing blood. The vascular endothelial cells
typically express receptors for VEGF, stain for PE-CAM, VE-CAM and
von Willebrand factor. The vascular endothelial cells produced by
the methods of the present invention would be useful as models for
genetically based vascular disease. An example could be human
hereditary telangiectasia, where patients have mutations in
TGF-.beta. receptors and a chronic bleeding syndrome. It is
difficult to isolate and maintain long-term cells from patients
with this disease to understand the pathology. Therefore,
genetically modified stem cells induced to differentiate to
vascular endothelial cells can provide a useful culture model. In
addition, the vascular endothelial cells produced by the present
methods can be used for transplantation and/or a route for delivery
of gene therapy.
[0105] In another aspect of the present invention there is provided
a method of treating or preventing a vascular disease in vascular
tissue, said method comprising: [0106] introducing to the vascular
tissue, a vascular endothelial cell and/or a vascular endothelial
progenitor cell that has been co-cultured in the presence of an
embryonic ectoderm and/or endoderm cell and/or extracellular medium
of an embryonic ectoderm and/or endoderm cell.
[0107] Preferably the vascular endothelial cells have been
differentiated from a stem cell co-cultured under conditions
described above.
[0108] Preferably, the vascular disease is caused by any one of
hereditary hemorrhagic telangiectasia, vascular deterioration as a
result of diabetes, or smoking.
[0109] In yet another aspect of the invention, there is provided a
differentiated cell produced according to the methods as
hereinbefore described. Preferably, the differentiated cell is a
cardiomyocyte, skeletal muscle cell, vascular endothelial cell or a
haematopoietic cell. The present invention also provides
differentiated cells produced according to the methods of the
invention that may be used for transplantation, cell therapy or
gene therapy. Preferably, the invention provides a differentiated
cell produced according to the methods of the invention that may be
used for therapeutic purposes, such as in methods of restoring
cardiac function in a subject suffering from a heart disease or
condition.
[0110] The differentiated cells may be used as a source for
isolation or identification of novel gene products including but
not limited to growth factors, differentiation factors or factors
controlling tissue regeneration, or they may be used for the
generation of antibodies against novel epitopes.
[0111] The differentiated cells produced according to the methods
of the present invention may be clonally expanded. A specific
differentiated cell type can be selectively cultivated from a
mixture of other cell types and subsequently propagated. Specific
differentiated cell types that are clonally expanded can be useful
for various applications such as the production of sufficient cells
for transplantation therapy, for the production of sufficient RNA
for gene discovery studies etc. The differentiated cells may be
used to establish cell lines according to conventional methods.
[0112] The differentiated cells produced according to the methods
of the present invention may be genetically modified. For instance,
a genetic construct may be inserted to a differentiated cell at any
stage of cultivation. The genetically modified cell may be used
after transplantation to carry and express genes in target organs
in the course of gene therapy.
[0113] The differentiated cells produced according to the methods
of the present invention may be preserved or maintained by any
methods suitable for storage of biological material. Effective
preservation of differentiated cells is highly important as it
allows for continued storage of the cells for multiple future
usage. Traditional slow freezing methods, commonly utilised for the
cryo-preservation of cell lines, may be used to cryo-preserve
differentiated cells.
[0114] The present invention further provides a cell composition
including a differentiated cell produced by the method of the
present invention, and a carrier. The carrier may be any
physiologically acceptable carrier that maintains the cells. It may
be PBS or other minimum essential medium known to those skilled in
the field. The cell composition of the present invention can be
used for biological analysis or medical purposes, such as
transplantation.
[0115] The cell composition of the present invention can be used in
methods of repairing or treating diseases or conditions, such as
cardiac disease or where tissue damage has occurred. The treatment
may include, but is not limited to, the administration of cells or
cell compositions (either as partly or fully differentiated) into
patients. These cells or cell compositions would result in reversal
of the condition via the restoration of function as previously
disclosed above through the use of animal models.
[0116] Throughout the description and claims of this specification,
the word "comprise" and variations of the word, such as
"comprising" and "comprises", is not intended to exclude other
additives, components, integers or steps.
[0117] The discussion of documents, acts, materials, devices,
articles and the like is included in this specification solely for
the purpose of providing a context for the present invention. It is
not suggested or represented that any or all of these matters
formed part of the prior art base or were common general knowledge
in the field relevant to the present invention as it existed in
Australia before the priority date of each claim of this
application.
[0118] The present invention will now be more fully described with
reference to the accompanying examples 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.
EXAMPLES
Example 1
Differentiation of Human Embryonic Stem (hES) Cells into
Cardiomyocytes
[0119] (a) Co-Culturing of hES Cells with END-2 Cells
[0120] Human embryonic stem cells (hES cells) Reubinoff et al,
Nature Biotech. 16:399-404) were co-cultured with END-2 cells
((Mummery et al, 1985, Dev Biol. 109:402-410)). The END-2 cells are
grown routinely in a 1:1 ratio of Dulbecco's minimum essential
medium (DMEM) and Ham's F12 medium (DF) with 7.5% FCS. Cells were
then passaged twice weekly, 1:5 using trypsin/EDTA (0.125% w/v; 50
mM resp). The hES cells were cultured in DMEM with 20% FCS, 0.1 mM
.beta.-mrcaptoethanol, 1% non-essential amino acids, 2 mM glutamine
plus antibiotics (pen/strep) on mitomycin (10 .mu.g/ml) treated
embryonic feeder cells. HES were subcultured by treating with
dispase and mechanical slicing of individual colonies into 6-10
pieces followed by transfer of the pieces to new feeder cells.
[0121] To initiate co-cultures, confluent cultures of END-2 cells
were first passaged 1:10 on to gelatin-coated glass coverslips or
tissue culture plastic wells in DMEM with 7.5% FCS and grown for 3
days to confluency. Monolayers were then treated with 10 .mu.g/ml
mitomycin C for 3 hours, washed 3 times in phosphate-buffered
saline (PBS) without Ca.sup.2+ and Mg.sup.2+, and hES medium added.
The hES cells were then dispersed using 10 mg/ml dispase for 3-5
minutes, followed by gentle agitation in a pipette to yield a
suspension containing small cell clumps of approximately 10-50
cells. These suspensions were then plated on the mitogenically
inactive END-2 monolayers, for 2-3 weeks, in hES complete medium.
The medium was replaced every 5-6 days with fresh hES medium.
(b) Co-Culturing of hES Cells with Visceral Endoderm Cells
[0122] Visceral endoderm cells were isolated from the three germ
layers of gastrulating mouse embryos at E7.5 (where E0.5 is noon on
the day of the vaginal plug, as described previously using dispase
(Roe/en et al, 1994, Dev. Biol. 166:716-728). The separated germ
layers were plated on to poly-L-lysine coated culture dishes in M16
medium and allowed to attach overnight. The next day, M16 was
replaced by hES complete medium, and on day 3 after germ cell
isolation, pieces of undifferentiated cell "transfers" were plated
on to the attached endodermal and ectodermal cell from the mouse
embryo. Cultures were then grown for 2 to 3 weeks and medium
refreshed every 5-6 days.
(c) Analysis of Co-Culture Experiments (a) and (b)
[0123] The cultures described in (a) and (b) above were scored for
the presence of areas of beating muscle from 10 days onwards.
(i) Immunofluorescence.
[0124] The cultures were then fixed after areas of beating muscle
became evident in 2% paraformaldehyde for 30 min, washed 3 times
and stored in PBS until use. The cultures were then stained to
verify the muscle phenotype using .alpha.-actinin antibodies
(monoclonal anti-.alpha.-actinin (sarcomeric) clone EA-53, dilution
1:1000, Sigma; secondary antibody: goat antimouse-IgG-Cy3).
(d) Results of Co-Culture Experiments (a) and (b)
[0125] (i) hES-END-2 Co-Culture.
[0126] During the first week of co-culture, the clumps of cells
gradually spread and differentiate to cells with mixed morphology
but with a relatively high proportion of epithelial-like cells. By
the second week, these swell to fluid-filled cysts (see FIG. 1A).
Between these, distinct patches of cells become evident which begin
to beat a few days later. Between 12 and 21 days, increasingly more
of these beating patches appear. There is no apparent difference
between glass and tissue culture substrates, both yielding beating
muscle as demonstrated in 3 independent experiments that indicated
15-20% of the wells containing one or more areas of beating muscle.
Beating rate is approximately 60 beats per minute and highly
temperature sensitive. These cells stain positively with
.alpha.-actinin, confirming that they are indeed muscle cells (FIG.
2).
(ii) hES-E7.5 Endoderm Co-Culture.
[0127] During the first week of culture, the hES pieces placed on
top of the endoderm gradually begin to spread and flatten and on
day 12, the first areas of beating muscle cells become evident.
This is not accompanied by the extensive cyst formation observed in
the END-2 co-cultures but areas resembling vascular endothelial
cell networks do appear at the edges of the culture.
Example 2
Differentiation of Human Embryonic Stem (hES) Cells into Skeletal
Muscle Cells
[0128] Human stem cells (hES) as used in Example 1 were placed on
ectoderm isolated from E7.5 day embryos (E0.5 is day of plug). With
sharpened tweezers and tungsten needles, the embryos were prepared
out of the decidua and kept on ice in HEPES-buffered DMEM
containing 10% FCS. After removing Reichert's membrane, the
embryonic and extraembryonic parts of the conceptus were separated
with tungsten needles. The node and primitive streak were removed
and the embryonic part incubated in 2.5% pancreatin and 0.5%
trypsin in PBS on ice for 8 min. After incubation, the embryos were
transferred to HEPES-buffered DMEM containing 10% FCS on ice. The
ectoderm, endoderm and mesoderm could then be cleanly isolated
using tungsten needles.
[0129] The hES cells initially resemble those on endoderm but by
day 18 there are areas of highly elongated, twitching cells that
resemble skeletal muscle. There are no areas reminiscent of beating
cardiac muscle although vascular networks (Vascular endothelial
cells) are evident.
Example 3
Differentiation of Human Embryonic Stem (hES) Cells into Vascular
Endothelial Cells
[0130] Human stem cells (hES) as used in Example 1 were placed on
ectoderm and/or endoderm cells. Co-culture condition of hES with
ectoderm or endoderm were as above. Vascular endothelial cells in
networks accompanied differentiation to other somatic cell
types.
Example 4
Co-Culture of Visceral Endoderm Cells and hES and Differentiated
Cardiomyocytes
a) Co-Culture
[0131] END-2 cells, P19 EC, hEC and hES cells were cultured as
described previously (Mummery et al. 1985, 1991; vanden Eijnden-van
Raaij et al. 1991; Slager et al. 1993; Reubinof et al. 2000). The
hES2 cell line from ESI (Reubinof et al. 2000) was used in all
experiments. To initiate co-cultures, mitogenically inactive END-2
cell cultures, treated for 1 h with mitomycin C (10 .mu.g
ml.sup.-1), as described in Example 1, replaced mouse embryonic
fibroblasts (MEFs) as feeders for hEC, mES and hES. Co-cultures
with P19EC, which are feeder independent, were initiated and
maintained as described previously (Mummery et al. 1991). Cultures
were then grown for 2-3 weeks and scored for the presence of areas
of beating muscle from 5 days onwards.
(b) Isolation of Primary Human Adult Cardiac Cells
[0132] Human atrial cells from surgical biopsies served as a
control for antibody staining, electrophysiology and
characterization of ion channels by RT-PCR. Cardiac tissue was
obtained with consent from patients undergoing cardiac surgery.
Atrial appendages routinely removed during surgery were immediately
transferred to ice cold Krebs-Ringer (KR) saline solution. Tissues
were trimmed of excess connective and adipose tissue and washed
twice with sterile KR solution. Myocardial tissue was minced with
sterile scissors, then dissociated to release individual cells by a
three-step enzymatic isolation procedure using published methods
(Peeters et al. (1995), Am. J. Physiol. 268, H1757-H1764). The
first step involved a 15-min incubation with 4.0 U mL.sup.-1
protease type XXIV (Sigma, St Louis, Mo., USA) at 37.degree. C.
Tissues were then transferred to a solution consisting of
collagenase 1.0 mg mL.sup.-1 and hyaluronidase 0.5 mg mL.sup.-1,
followed by three further incubations with collagenase (1.0 mg
mL.sup.-1) for 20 min each at 37.degree. C. Tissue extracts were
combined and the calcium concentration restored to 1.79 mmol
L.sup.-1, Cardiomyocytes were transferred to tissue culture medium
M199 enriched with 10% FBS, penicillin (100 U
mL.sup.-1)/streptomyocin (100 .mu.g mL.sup.-1), 2.0 mmol L.sup.-1
L-carnitine, 5.0 mmol L.sup.-1 creatine, 5.0 mmol L.sup.-1 taurine
and seeded directly on to glass cover-slips coated with 50 .mu.g
mL.sup.-1 poly L-lysine and cultured overnight.
(c) Immunocytochemistry
[0133] Attached primary cardiomyocytes, mES (E14 and R1) and
hES-derived cardiomyocytes were fixed with 3.0% paraformaldehyde in
PBS with Ca.sup.2+ and Mg.sup.2+ for 30 min at room temperature,
then permeabilized with 0.1% triton X 100 in PBS for 4 min.
Immunocytochemistry was performed by standard methods using
monoclonal antibodies directed against sarcomeric proteins
including .alpha.-actinin and tropomyosin (Sigma). Antibodies
specific for isoforms of myosin light chain (MLC2a/2v) were used to
distinguish between atrial and ventricular cells (gift of Dr Ken
Chien) (Table 1). Secondary antibodies were from Jackson
lmmunoresearch Laboratories. Cultured cardiac fibroblasts served as
a negative control for sarcomeric proteins and cells were
visualized using a Zeiss Axiovert 135M epifluorescence microscope
(Carl Zeiss, Jena GmbH, Germany). Images were pseudocoloured using
image processing software.
TABLE-US-00001 TABLE 1 Antibodies used to stain atria
cardiomyocytes Primary antibody Dilution Secondary antibody
Dilution Mouse anti-.alpha.-actinin IgG 1:800 Goat anti-mouse 1:250
IgG-cy3/FITC conjugated Mouse anti-tropomyosin IgG 1:50 Goat
anti-mouse 1:250 IgG-cy3 conjugated Polyclonal rabbit anti-mouse
1:500 Goat anti-rabbit 1:250 mic-2a (atrial) IgG-cy3 conjugated
Hoechst (nucleic acid) 1:500
(d) Semi-Quantitative RT-PCR for Ion Channel Expression
[0134] P19EC cells were differentiated into beating muscle by the
aggregation protocol in the presence of 1% dimethyl sulphoxide
(Rudnicki & McBurney, 1987). After 16 days in these culture
conditions, beating areas were excised and RNA was isolated using
Trizol (Gibco) andreversed transcribed using M-MLV-RT (Gibco).
Primers for cardiac actin (Lanson et al. (1992) Circulation 85,
1835-1841), MLC2v (Meyer et al. (2000) FEBS Lett, 478, 151-158),
ERG (Lees-Miller et al. (1997), Circ. Res. 81, 719-726) and Kir2.1
(Vandorpe et al. (1998), J. Biol. Chem. 273, 21542-21553) were used
as described previously. Primers for mouse L-type calcium channel
subunit .alpha.1c (sense 5-CCAGATGAGACCCGCAGCGTAA; antisense
5'-GTCTGCGGCGTTCTCCATCTC; GenBank accession no. L01776; product
size 745 bp), Scn5a (sense 5'-CTTGGCCAAGATCAACCTGCTCT; antisense
5'-CGGACAGGGCCAAATACTCAATG; AJ271477; 770 bp) and .beta.-tubulin
(sense 5-TCACTGTGCCTGAACTTACC; antisense 5'-GAACATAGCCGTAAACTGC;
X04663; 319 bp) were designed using Vector NTI software (InforMax,
North Bethesda, Md., USA).
(e) Patchclamp Electrophysiology
[0135] Experiments were performed at 33.degree. C., using the whole
cell voltage clamp configuration of the patch-clamp technique.
After establishment of the gig a seal the action potentials were
measured in the current clamp mode. The data were recorded from
cells in spontaneously beating areas using an Axopatch 200B
amplifier (Axon Instruments Inc., Foster City, Calif., USA). Output
signals were digitized at 2 kHz using a Pentium III equipped with
an AD/DAC LAB PC+ acquisition board (National Instruments, Austin,
Tex., USA). Patch pipettes with a resistance between 2 and 4 M
.OMEGA. were used. Composition of the bathing medium was 140 mM
NaCl, 5 mM KCl, 2 mM CaCl.sub.2, 10 mM HEPES, adjusted to pH 7.45
with NaOH. Pipette composition: 145 mM KCl, 5 mM NaCl, 2 mM
CaCl.sub.2, 10 mM EGTA, 2 mM MgCl.sub.2, 10 mM HEPES, adjusted to
pH 7.30 with KOH.
(f) Results of Co-Culturing
[0136] (i) mEC-END-2 Co-Cultures
[0137] Two days after initiation of co-cultures with END-2 cells,
P19 EC cells aggregated spontaneously and 7-10 days later many of
the aggregates contained areas of beating muscle (FIG. 3a), as
described previously (Mummery et al. 1991). Electrophysiology and
RT-PCR showed that functional ion channels characteristic of
embryonic cardiomyocytes were expressed in these cells (FIG. 4,
Table 2).
TABLE-US-00002 TABLE 2 Relative levels of cardiac marker and ion
channel mRNA expression as determined by semiquantitative RT-PCR.
Identical amounts of cDNA of undifferentiated P19 (EC),
differentiated P19 cardiomyocytes (CMC) and adult mouse heart
(Heart) were PCR amplified for the indicated gene products.
Relative levels for each product are indicated. EC CMC Heart Ion
channel and current Cardiac actin + ++ +++++ MLC2v + +++ +++++
.alpha.1c + +++ ++++ L-type calcium channel, I.sub.Ca Scn5a + +
+++++ Heart specific sodium channel, I.sub.Na ERG ++ ++ ++++
Delayed rectifier potassium channel, I.sub.Kr Kir2.1 - + ++++
Voltage-gated potassium channel, I.sub.K1 Tubulin +++ +++ +++
(ii) mES-END-2 Co-Cultures
[0138] Two independent mouse ES cell lines (E14 and R1) were tested
for their response to co-culture conditions. Although the cultures
were not initiated as single cell suspensions, within 3 days larger
aggregates than initially seeded were evident for both cell lines
(FIG. 3b,c). Almost simultaneously, extensive areas of
spontaneously beating cardiomyocytes were evident in the R1 ES cell
cultures, although, only 7 days later, (smaller) areas of beating
muscle were found in the E14 ES cells. Cells in beating areas
exhibited the characteristic sarcomeric banding pattern of myocytes
when stained with .alpha.-actinin (see FIG. 6d).
(iii) hEC- and END-2 Co-Cultures
[0139] The human EC cell line GCT27X is a feeder-dependent,
pluripotent EC cell line, with characteristics similar to human ES
cells (Pera et al. (1989), Differentiation 42, 10-23). In
co-culture with END-2 cells, formation of large aggregates was
observed (FIG. 3d). However even after 3 weeks, there was no
evidence of beating muscle.
(iv) hES-END-2 Co-Cultures
[0140] During the first week of co-culture, the small aggregates of
cells gradually spread and differentiated to cells with mixed
morphology but with a relatively high proportion of epithelial-like
cells. By the second week, these swelled to fluid-filled cysts (not
shown). Between these, distinct patches of cells become evident
which begin to beat a few days later. Between 12 and 21 days, more
of these beating patches appear (e.g. FIG. 3e). Overall, 15-20% of
the wells contains one or more areas of beating muscle. Beating
rate is approximately 60 min.sup.-1 and is highly temperature
sensitive, compared with mouse ES-derived cardiomyocytes. These
cells stain positively with .alpha.-actinin, confirming their
muscle phenotype (FIG. 6e). In contrast to mES and P19EC-derived
cardiomyocytes, however, the sarcomeric banding patterns were
poorly defined but entirely comparable with primary human
cardiomycytes grown for only 2 days in culture (FIGS. 5 and 6a-c).
It is clear that while primary human cardiomycytes initially retain
the sarcomeric structure, standard culture conditions result in its
rapid deterioration (FIG. 5). It may be assumed that hES culture
conditions are not optimal for cardiomyocytes so that the
hES-derived cardiomyocytes similarly exhibit deterioration in their
characteristic phenotype. It will be essential to optimize these
conditions to obtain fully functional cardiomyocytes from stem
cells in culture. Despite deterioration in sarcomeric structure,
hES derived cardiomyocytes continued to beat rhythmically over
several weeks and action potentials were detectable by current
clamp electrophysiology (FIG. 4b), performed by inserting
electrodes into aggregates, as shown in FIG. 4(c). However,
carrying out electrophysiology in this manner, i.e. in aggregates
rather than single cells, yields action potentials that are the
accumulated effects of groups of cells. They are therefore
difficult to interpret and to attribute to either ventricular,
atrial or pacemaker cells. Work is currently in progress to
dissociate and replate aggregates to allow single cell
determinations.
(v) Cardiac Ion Channel Expression During Stem Cell
Differentiation
[0141] The order in which ion currents, responsible for the
subsequent phases of the adult action potential, appear during
heart development has been established in electrophysiological
studies (Davies et al. (1996), Circ. Res. 78, 15-25). Inward L-type
Ca.sup.2+ currents play a dominant role during early cardiac
embryogenesis, whereas inward Na.sup.2+ currents increase only just
before birth (Davies et al. 1996). Mouse ES and P19 EC cells
display similar timing in ion current expression (Wobus et al.
(1994), In Vitro Cell. De. Biol. 30A, 425-434). To unravel the
sequence of ion-channel expression at the molecular level during
differentiation of P19 EC cells, we performed RT-PCR on RNA
isolated from undifferentiated and 16-day-old beating clusters of
P19-derived cardiomyocytes and relatively positive resting membrane
potential between -40 and -60 mV (little to no IK1). These results
indicate that day 16 P19 cardiomyocytes resemble fetal
cardiomyocytes with respect to ion channel expression, as has been
described previously for mES-derived cardiomyocytes (Doevendans et
al. (1998), cardiovasc. Res. 39, 34-39).
[0142] The results of the work described here show that VE-like
cells induce/promote differentiation of pluripotent cells to
cardiomyocytes. These cells include pluripotent mouse EC cells,
mouse ES as well as human ES cells, which are demonstrated for the
first time to respond to inductive cues derived from cells similar
to those normally adjacent to the region of heart development in
the embryo.
Example 5
Cardiomyocyte Differentiation of Human Embryonic Stem Cells Induced
by Co-Culture with Visceral Endoderm-Like Cells
(a) Methods
i) Cell Culture
[0143] END-2 cells and hES cells were cultured as described in
Example 1. The hES2 cell line from ES Cell International Pte Ltd
was used in all experiments. To initiate co-cultures, mitogenically
inactive END-2 cell cultures, treated for 1 hr with mitomycin C (10
.mu.g/ml), replaced mouse embryonic fibroblasts (MEFs) as feeders
for hES cells. Co-cultures were then grown for up to 6 weeks and
scored for the presence of areas of beating muscle from 5 days
onwards. HepG2 cells, a carcinoma cell line resembling liver
parenchymal cells (Knowles et al, (1980) Science 209:497-9), were
cultured in DMEM plus 10% FCS and passaged twice weekly.
Co-cultures were initiated as for END-2 cells although HepG2 do not
grow as a confluent monolayer; hES cells attached preferentially to
groups of HepG2. Hanging-drop cultures of P19 EC cells were
initiated and maintained in DMEM/Ham's F12 (1:1), as described
previously (Mummery et al, 1991), using regular FCS while bulk
production of P19 embryoid bodies in the presence or absence of
END-2 conditioned medium for isolation of RNA for Northern
blotting, was in the presence of charcoal-stripped FCS, as
described (Mummery et al, 1991) to reduce background levels of
cardiomyocyte differentiation caused by lipophilic substances
possibly with retinoid-like activity in FCS. P19 clone 6 were
cultured in aMEM and treated aMEM conditioned by END-2 cells. For
electrophysiology, beating aggregates were dissociated using
collagenase and replated on gelatine-coated coverslips.
ii) Immunohistochemistry
[0144] Attached primary cardiomyocytes hES-derived cardiomyocytes
were fixed with 3.0% paraformaldehyde in PBS with Ca.sup.2+ and
Mg.sup.2+ for 30 minutes at room temperature, then permeablized
with 0.1% triton.times.100 in PBS for four minutes.
Immunocytochemistry was performed by standard methods using
monoclonal antibodies directed against sarcomeric proteins
including .alpha.-actinin and tropomyosin (Sigma). Antibodies
specific for isoforms of myosin light chain (MLC2a/2v) were used to
distinguish between atrial and ventricular cells (gift of Dr. Ken
Chien). Oct-4 and al c antibodies were from Sigma. Secondary
antibodies were from Jackson Immunoresearch Labs.
iii) Culture of Primary Adult and Fetal Cardiomyocytes.
[0145] Primary tissue was obtained from patients following standard
informed consent procedures and approval of the ethics committee of
the University Medical Centre, Utrecht. Adult cardiomycytes were
isolated and cultured, as described previously (Mummery et al,
(2002) J. Anat 200:233-242). Fetal cardiomycytes were isolated from
fetal hearts perfused by Langendorff and cultured in a similar way
with the exception the supernatant remaining after mild
centrifugation contained the majority of viable cardiomyocytes that
attached to laminin-coated coverslips. For electrophysiology, cells
were collected in Tyrode's buffer with low calcium (Sipido et al,
1998. Cardiovasc. Res. 37:478-88) and replated directly in glass
wells for patch-clamp.
iv) (Semi-Quantitative) RT-PCR
[0146] RNA was isolated using Trizol (Gibco) and reversed
transcribed using M-MLV-RT (Gibco). Primers for .alpha.1c (Van
Gelder et al., 1999), Kv4.3 (Calmels et al., 2001, Ref) and ANF
(Kehat et al, 2001, J. Clin. Invest. 108, 407-414) were as
described. Primers for human KvLQT1 (sense 5'-TTCTTGGCTCGGGGTTTGCC;
antisense 5'-TGTTGCTGCCGCGATCCTTG; GenBank accession no. AF000571;
product size 723 bp) and .beta.-tubulin (sense
5'-TGGCTTTGCCCCTCTCACCA; antisense
5'-CGGCGGAACATGGCAGTGAA;AF141349; 369 bp) were designed using
VectorNTI software (InforMax, North Bethesda, USA). Annealing
temperatures and number of amplification cycles were as follows:
ANF, 58.degree. C., 35 cycles; .alpha.1c, 56.degree. C., 38 cycles;
Kv4.3 55.degree. C., 35 cycles; KvLQT1, 58.degree. C., 32 cycles;
.beta.-tubulin, 61.degree. C., 30 cycles. Products were analyzed on
an ethidium bromide stained 1.5% agarose gel.
v) Northern Blotting.
[0147] RNA was isolated from P19 aggregates and poly A+RNA
selected, as described previously (Mummery et al, 1990, Dev. Biol.
142: 406-413).
vi) Electrophysiology
[0148] Experiments were performed at 33.degree. C., using the whole
cell voltage clamp configuration of the patch clamp technique.
After establishment of the gigaseal, action potentials were
measured in the current clamp mode. Data were recorded from cells
in spontaneously beating areas using an Axopatch 200B amplifier
(Axon Instruments Inc., Foster City, Calif., U.S.A.). Output
signals were digitized at 2 kHz using a Pentium III equipped with
an AD/DAC LAB PC+ acquisition board (National Instruments, Austin,
Tex., U.S.A.). Patch pipettes with a resistance between 2 and 4 MO
were used. Composition of the bathing medium was 140 mM NaCl, 5 mM
KCL, 2 mM CaCL.sub.2, 10 mM HEPES, adjusted to pH 7.45 with NaOH.
Pipette composition: 145 mM KCL, 5 mM NaCL, 2 mM CaCL.sub.2, 10 mM
EGTA, 2 mM MgCL.sub.2, 10 mM HEPES, adjusted to pH 7.30 with
KOH.
(b) Cardiomyocyte Differentiation of Human ES Cells.
[0149] The majority of the experiments described here were carried
out using the hES2 cell line (Reubinoff et al, 2000). The cells
were maintained in an undifferentiated state by co-culture with
mitomycin C-treated MEF "feeder cells" in serum-containing medium,
as described (Mummery et al, 2002); under these conditions, all
cells in the bulk of the culture showed nuclear staining for oct-4,
although any flattened cells at the edge of the culture were
negative. Oct-4 expression thus correlated with phenotypic
characteristics of undifferentiated cells. hES cells were
subcultured by transferring small clumps of undifferentiated cells
either to new, mit.C-treated MEFs or confluent cultures of
mit.C-treated END-2 cells. After approximately 5d under these
conditions, epithelial cells became evident which over the next few
days become fluid-filled cysts (FIG. 1A). These stain for
alphafetoprotein, suggesting that they represent extraembryonic
visceral endoderm. In addition, by 10 d areas of rhythmically
contracting cells in more solid aggregates become evident (FIG.
1A,3e) with a variety of overall morphologies (FIG. 1B,3e). 16-35%
of wells in a 12-well plate contain beating areas each of which can
be dissociated and replated to yield up to 12 new colonies of
beating cells with a 2-D rather than 3-D morphology; this
facilitates access to the cells for further characterization by
patch-clamp electrophysiology (FIG. 4,FIG. 9; see below). Both
before and after dissociation, hES-derived cardiomycytes beat 45-60
times per minute, sometimes irregularly; beating was upregulated in
response to pharmacological agonists such as carbachol,
isoprenaline and phenylephrine.
[0150] In order to characterize the cardiomycytes further,
immunofluorescent staining was carried out for a number of
sarcomeric proteins, BIDOPY-ryonadine was used as a vital stain for
sarcomeres and the expression of ion channels analysed by RT-PCR
(FIG. 8). In each case, primary human fetal (17 weeks) and adult
atrial and/or ventricular tissue was used as a controlled
reference. The data showed that hES-derived cardiomyocytes exhibit
sarcomeric striations when stained with .alpha.-actinin, organized
in separated bundles (FIG. 2). These are reminiscent of the bundles
observed in fetal cardiomyocytes, although the individual
sarcomeres are less well defined, but are quite different from the
highly organized, parallel bundles observed in ventricular cells
from biopsies of adult human heart.
(c) Expression of Cardiac Ion Channels and Stem Cell/Sarcomere
Markers in hES/END-2 Co-Cultures.
[0151] Expression of cardiac specific ion channels was determined
in undifferentiated hES cells and in differentiating cells 9 and
14-days after initiation of co-culture with END-2 cells (FIG. 8).
As shown by others previously (Kehat et al., 2001), areas of
beating hES-derived cardiomyocytes express ANF. Expression of the
.alpha.-subunits of the cardiac specific L-type calcium channel
(.alpha.1c) and the transient outward potassium channel (Kv4.3) are
also detected, the expression of Kv4.3 preceding commencement of
beating by several days. RNA for the delayed rectifier potassium
channel KvLQT1 is found in undifferentiated cells, it disappears
during early differentiation and reappears at somewhat later
stages.
(d) Electrophysiology
[0152] Patch clamp electrophysiology on dissociated, replated
aggregates of hES cardiomyocytes showed that range of (electrical)
phenotypes were present in the cultures (FIG. 9A) that were
comparable with primary human cardiomyocytes of fetal origin (FIG.
9B). Ventricular-like action potentials predominated (28 of 31
determinations) but both atrial and pacemaker-like cells were also
present (2 and 1 of 31 determinations, respectively). Of note was
the relatively slow upstroke velocity (7.0+/-0.8 V/s) and low
membrane potential (FIG. 9A), indicating that cells were relatively
immature even compared with fetal human cardiomyocytes of 17 weeks
gestation. In areas of co-culture in which the cells were not
beating but had adapted morphologies indistinguishable from beating
areas, current injection was sufficient to induce repeated action
potentials and sustained rhythmic contractions. In addition
co-cultures of hES cells with HepG2 cells resulted cardiomyocytes
with action potentials similar to those in hES-END-2
co-cultures.
[0153] Calcium transients in groups of beating hES cardiomyocytes
were also determined in real time using Fura-2, for which the
fluorescence emission spectrum depends on the intracellular calcium
concentration. FIG. 9B shows that repetitive Ca.sup.2+ transients
are generated in hES cardiomyocytes, reflecting their ability to
beat in the absence of obvious conductance cells.
(e) Effects of END-2 Conditioned Medium on Aggregates of P19EC
Cells.
[0154] We have shown previously (Mummery et al, 1991) that medium
conditioned for 24 h by END-2 cells can induce their
differentiation so that within 10 days nearly all aggregates
replated after 3d growth as hanging drops, contain beating muscle.
In the presence of regular FCS, differentiation is significantly
enhanced above background levels. Background differentiation levels
are reduced virtually to zero if the FCS is passed over an
activated charcoal column (DCC-FCS) to remove residual retinoids;
in addition, differentiation is blocked by activin (van den
Eijnden-van Raaij et al, 1991, Mech. Dev. 33:157-166). END-2-CM was
tested on both hES and mES. cells cultured on MEFs (without MEFs
the cells die/differentiate in a non-directed way) but failed to
override any differentiation inhibiting activity secreted by MEFs.
The P19EC assay system was preferred because these cells are feeder
independent for undifferentiated growth. These experiments show
that the END-2 CM has a dose dependent effect in promoting the
appearance of cardiomyocytes in replated aggregates in two
independent clones of P19 EC cells (FIG. 10A). More importantly,
the effect appears to be related to an early effect on the
differentiation of nascent mesoderm; Northern blot analysis shows
that END-2 CM upregulates an early mesoderm marker Brachyury T
(Hermann, 1991) in the aggregation phase during the first 3 days
(FIG. 10B), that levels are maintained immediately after plating
but that after 9 days, Brachyury T is no longer detectable.
Moreover, the induction of Brachyury T is blocked by the additional
presence of activin (FIG. 10B), consistent with its ability to
block the formation of beating muscle in aggregates. The transient
expression of Brachyury T is similar to that observed during early
mesoderm differentiation during gastrulation of the mouse
embryo.
[0155] Control of sustained growth and the ability to induce
specific differentiation pathways are essential if human embryonic
stem (hES) cells are to reach their potential in the treatment of
disease by cell transplantation therapy.
[0156] IT is therefore shown that co-culture of hES cells with
visceral endoderm-like cells from the mouse initiates a
differentiation programme that leads to the formation of beating
muscle cells. Expression of sarcomeric marker proteins and ion
channels demonstrates these cells are cardiomyocytes, while
patch-clamp electrophysiology on single cells demonstrates that the
majority is ventricular in phenotype. This system provides a model
for the study of human cardiomyocytes in culture, generally
difficult to achieve, and perspectives for cardiomyocytes
transplantation therapies where it is envisaged that replacement of
ventricular cells lost in ischemic heart disease will help restore
cardiac function. This is the first demonstration of the induction
of cardiomyocyte differentiation in a hES cell line that does not
undergo spontaneous differentiation to somatic lineages.
Example 6
Myocardial Infarction Model in Mice
[0157] In order to test the ability of stem-cell-derived
cardiomyocytes to restore cardiac function, a MI model has been
developed in mice. In pentobarbital anesthetized adult mice, the
chest is opened through a midsternal approach. The anterior
descending branchis identified and ligated. Successful procedures
induce a discoloration of the distal myocardium. The chest is
closed with three sutures and the animal is allowed to recover. In
total, 17 animals have been operated on. Seven received a sham
procedure including positioning of the suture and 10 were ligated.
Four weeks after MI the mice were anaesthetized again using the
same medication by intraperitoneal injection. For the haemodynamic
study the animals were incubated, and connected to a rodent
respirator (Hugo Sachs Electronics, March-Hugstetten Germany).
Instrumentation was performed with the chest closed by introducing
a catheter into the jugular vein.
[0158] A 1.4 French conductance-micromanometer (Millar Instruments,
Houston, Tex., USA) was delivered to the left ventricle through the
carotid artery. Pressure and conductance measurements were recorded
using Sigma SA electronic equipment (CDLeycom, Zoetermeer, the
Netherlands) and stored for offline analysis. A typical pressure
volume (PV) loop recorded in a normal heart is presented in FIG.
7(a). From the PV-loops many haemodynamic parameters can be deduced
including the end-systolic PV relationship (ESPVR) and preload
recruitable stroke work (PRSVV).
[0159] Finally, the invention as hereinbefore described is
susceptible to variations, modifications and/or additions other
than those specifically described and it is understood that the
invention includes all such variations, modifications and/or
additions which may be made it is to be understood that various
other modifications and/or additions which fall within the scope of
the description as hereinbefore described.
Sequence CWU 1
1
10122DNAArtificial Sequenceoligonucleotide primer 1ccagatgaga
cccgcagcgt aa 22221DNAArtificial Sequenceoligonucleotide primer
2gtctgcggcg ttctccatct c 21323DNAArtificial Sequenceoligonucleotide
primer 3cttggccaag atcaacctgc tct 23423DNAArtificial
Sequenceoligonucleotide primer 4cggacagggc caaatactca atg
23520DNAArtificial Sequenceoligonucleotide primer 5tcactgtgcc
tgaacttacc 20619DNAArtificial Sequenceoligonucleotide primer
6gaacatagcc gtaaactgc 19720DNAArtificial Sequenceoligonucleotide
primer 7ttcttggctc ggggtttgcc 20820DNAArtificial
Sequenceoligonucleotide primer 8tgttgctgcc gcgatccttg
20920DNAArtificial Sequenceoligonucleotide primer 9tggctttgcc
cctctcacca 201020DNAArtificial Sequenceoligonucleotide primer
10cggcggaaca tggcagtgaa 20
* * * * *