Transcript Heart muscle cells - Bio-Link

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Directed differentiation of ES cells
into cardiac mesoderm
Directed differentiation allows
production of a desired cell type
Pluripotent
Multipotent
Ectodermal cell
ES cell
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Differentiated
cells
Heart muscle cells (or
cardiomyocytes) derived by
this process can be used for:
brain
Mesodermal cell
heart
Endodermal cell
pancreas
1. replacement of damaged
tissue (e.g. following a heart
attack).
2. development of new therapies
to treat chronic
abnormalities of heart
function (e.g. arrhythmia)
by studying individual
beating heart muscle cells
in a dish.
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Diseases of heart muscle tissue
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Cardiac muscle
– Tissue composed of individual cells, unlike skeletal
myofibers
with many nuclei
– Cells coupled by gap junctions to enable rapid spread of
electrical signals
– Cells joined end-to-end by adhesive discs
Diseases of cardiac muscles
– Congestive heart failure, or ineffective pumping due to
cardiomyocyte dysfunction, affects 4.8 million people in U.S.
–Heart attack, also known as myocardial infarction
–Heart arrhythmias, or abnormal activity (palpitations)
– 1% of live births display a congenital heart defect
A four-chambered heart underlies
double circulation (birds & mammals)
•
This system is the first
functional unit that forms in
the embryo.
•
Circulation provides
nourishment to organs
(nutrients and oxygen).
•
The heart is the main organ
that pumps blood through the
blood vessels.
•
The heart contains four
chambers in birds and
mammals (simpler in fish &
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amphibians).
Left atrium
Left ventricle
Heart
Blood vessels
Blood cells
•
Atrioventricular
valves
Right ventricle
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The circulatory system is:
–
–
–
Right atrium
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Heart muscle, to perform its unique function,
is different from skeletal and smooth muscle
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1. Cardiac muscle cells:
Three types of muscle cells
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Have visible myofibrils
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Occur only in heart
•
Involuntary movement
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Cells joined by intercalating disks to allow
rapid spread of electrical signals
2. Skeletal muscle cells:
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Elongated multinucleate cells called
myofibrils.
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Voluntary movements
3. Smooth muscle cells:
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Individual, spindle-shaped mononuclear
cells
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Found in gut, blood vessels and ducts of
glands
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Involuntary movements
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How are cardiomyocytes generated
during development?
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Lineage restrictions
Pluripotent
Multipotent
Differentiation
Ectodermal cell
?
ES cell
Mesodermal cell
Endodermal cell
?
Multipotent
cardiovascular
progenitor
Cardiomyocyte
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Heart, blood and blood vessels are
derived from the lateral mesoderm
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Heart, circulatory
system, smooth
muscle
Skeletal muscles
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Formation of heart tube from
germ layers (in the chick)
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Cardiac looping and chamber formation
in the human embryo
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Secreted factors specify
heart-forming mesoderm in vivo
Lineage restrictions
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Differentiation
FGF8
Anterior lateral plate
mesodermal cell
Cerberus
BMPs
Wnts
Lateral plate
mesodermal cell
Cardiogenic
mesodermal
cell
Cardiomyocyte
Noggin
Posterior lateral plate
mesodermal cell
Hemangiogenic
mesodermal cell
Blood cells
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Distinct cell signaling pathways induce
cardiomyocyte-specific genes
Cell signaling pathway
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Gene expression
Heart muscle cell
(Cardiomyocyte)
BMPs
BMP-RII
Progenitor
cell
OFF
OFF
Heart muscle cell
(Cardiomyocyte)
ON
Myosin light chain 2 gene
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Markers of the cardiomyocyte
progenitor lineage
Cardiomyocyte lineage
Bry
Nkx2.5+ / Isl-1+
Cardiogenic
mesodermal
progenitor
Nkx2.5+ / Isl-1+/ GATA4+/ Flk-1+
Multipotent
cardiovascular
progenitor
(2nd heart field)
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Differentiation
Nkx2.5+ / c-Kit+
1st heart field
progenitor
Mesodermal
Precursor
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Left ventricle
Nkx2.5+ / Isl-1+/ GATA4+
Cardiac
progenitor
Remainder of heart
Isl-1+/ Flk-1+
Vascular
progenitor
Blood vessels
and blood cells
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Markers of the mature
cardiomyocyte lineage
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Sources of cells for cardiomyocyte
replacement therapies: the dish
Directed
differentiation
Transplantation &
engraftment
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Stable formation
of mural grafts
Sources of cells for cardiomyocyte
replacement therapies: the heart
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Directed differentiation protocol for
mouse ES cells into cardiomyocytes
- LIF
+ serum
mES cells
- LIF
+ serum
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- LIF
+ serum
Day 5-7
Nkx2.5+
progenitors
Day 2
EBs
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Day 7-10
beating
cardiomyocytes
Wnt/BMP/Activin and serum are often used to induce cardiomyocyte precursors
ES cells
2 - 4 days
4 days
2 days
EBs
Nkx2.5+
cardiomyogenic
progenitors
Beating
cardiomyocytes
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Directed differentiation of mouse ES cells
produces vascular and cardiac lineages
Isl-1+/ Flk-1+
Vascular
progenitors
Fraction of Flk-1+
progenitors at day 3-5
%
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Nkx2.5+ / Isl-1+/ Gata4+
Cardiac
progenitors
Fraction of Nkx2.5+
progenitors at day 5
%
%
3.84 %
Nkx2.5::eGFP
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Isolated heart muscle cells
beat spontaneously
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Directed differentiation of
ES cells creates specialized
beating heart cells in vitro.
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How do heart muscle cells
(or cardiomyocytes) beat in
a dish?
1. Express cardiac troponin T
(cTnT).
2. Some cells become node cells
(HCN4+) that beat
spontaneously.
3. These cells express Ca++ ion
channels that confer
contractile properties.
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Directed differentiation protocol for
human ES cells to cardiomyocytes
- bFGF
+ 20% FBS
- bFGF
+ 20% FBS
BMP-4
hES cells
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- bFGF
+ 20% FBS
Day 7
Nkx2.5+
progenitors
Day 4
hEBs
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Day 11
beating
cardiomyocytes
Wnt/BMP/Activin and serum are often used to induce cardiomyocyte precursors
4 days
hES cells
3 days
20% FBS
BMP-4
12.5-25 ng/ml
4 days
20% FBS
Human
embryoid
bodies
20% FBS
Nkx2.5+
cardiomyogenic
progenitors
Beating
cardiomyocytes
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Rodent model of heart attack to test
stem cell-based repair of heart tissue
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Sources for stem/progenitor
cells that might be used to
repair tissue following a heart
attack:
–
–
–
–
–
–
ES cells
Cardiac stem cells
Muscle stem cells
Mesenchymal cells from adult
bone marrow
Endothelial progenitors
Umbilical cord blood cells
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Rats, mice and pigs are
commonly used to test
whether cells can repair hearts
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Conditions mimicking those
of a heart attack are achieved
in animal models by ligation of
the coronary artery
Stem cells directly or indirectly
repair damaged heart tissue
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Summary
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• Directed differentiation of ES cells into heart muscle cells is the
production of beating cardiomyocytes in a dish using defined factors.
• The factors used are crucial for generating these muscle cells during
normal embryonic development.
• Formation of the mammalian heart involves complex tissue
rearrangements as well as precise cell differentiation.
• Similar experimental conditions lead to directed differentiation of
cardiomyocytes from either mouse or human ES cells.
• A wide variety of sources may provide cells that are effective for
repairing heart muscle tissue after is has been damaged due to a heart
attack.
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