Work Force Development: Directed Differentiation (Heart Muscle) Primer
Learning Items B, C, D, E, J, K, S, V
Essential questions (B, J and K)
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How can stem cells be induced to adopt various fates in a dish?
What are the potential therapeutic uses for differentiated stem cells?
How are cardiomyocytes specified during normal embryonic development?
What are some common diseases that affect heart muscle tissue?
How does our knowledge of heart development inform experimental
approaches to create cardiomyocytes in vitro from stem cells?
Key knowledge and skills you will acquire as a result of this lecture
Students will know:
 Key Terms: directed differentiation, embryoid body, pluripotent, multipotent,
unipotent, transfection, cardiomyocyte, infarction, arrhythmia, atrium,
ventricle, cardiac troponin T, lateral plate mesoderm, cardiovascular
 The developmental potential of various stem cell populations.
 Advantages and disadvantages of in vitro approaches to understand and treat
diseases of the heart.
 Cellular and genetic basis for some well-described cardiac diseases.
 Crucial developmental signaling molecules that are used to derive muscle
precursors and cardiomyocytes from ES cells in a dish.
Students will be able to:
 Describe how secreted factors present during normal development are
employed to direct differentiation of stem cells into cardiomyocytes.
 Compare and contrast the underlying mechanisms responsible for various
cardiac diseases.
 Discuss the potential pitfalls when using laboratory disease models to arrive
at new treatments for myocardial infarction and arrhythmia.
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ES cells from the inner cell mass can produce all three germ layers and are
pluripotent. Cells in each germ layer retain the ability to proliferate and will give
rise to a more restricted spectrum of cells. Therefore, they are multipotent cells.
During embryonic development, proliferating precursors or progenitors eventually
appear that have very limited fates and are unipotent. Stem cells or progenitors
thus undergo successive steps of lineage restriction that limit the eventual cell types
they can produce during development. The directed differentiation of ES cells in
culture is the process of targeted conversion of ES cells into specialized cells such as
heart muscle cells. These cells can be used for: a) replacement of damaged heart
tissue (e.g. following a heart attack); and b) development of new therapies to treat
chronic abnormalities of heart function (e.g. arrhythmia) by studying individual
beating heart muscle cells in a dish (Slide 1).
A large number of research laboratories have focused their efforts on
developing methods to direct differentiation of ES cells into cardiomyocytes, which
are the primary beating cells in the heart. These muscle cells are coupled by
junctions, including gap junctions, that allow electrical signals to spread very rapidly
from one cell to the other and thus synchronize muscle contraction throughout the
tissue. Cardiovascular disease (CVD), which includes hypertension, coronary heart
disease, stroke and congestive heart failure, has ranked as the primary cause of
death in the United States every year since 1900 (except in 1918 when the nation
struggled with an influenza epidemic). Nearly 2,600 Americans die of CVD each day,
roughly one person every 34 seconds. Given the aging of the population and
relatively dramatic recent increases in the prevalence of cardiovascular risk factors
such as obesity and Type 2 diabetes, CVD will be a significant health concern well
into the 21st century.
Cardiovascular disease can deprive heart tissue of oxygen, thereby killing
cardiac muscle cells (cardiomyocytes). This loss triggers a cascade of detrimental
events, including formation of scar tissue, an overload of blood flow and pressure
capacity and overstretching of the viable cardiac cells that attempt to sustain heart
output. This eventually leads to heart failure and death. Restoring damaged heart
muscle tissue, through repair or regeneration, is therefore a new strategy with great
potential to treat heart failure.
There are a number of diseases that affect heart muscle. 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, is the
interruption of blood supply to a part of the heart, which causes heart cells to die.
This is most commonly due to occlusion (blockage) of a coronary artery after
rupture of a vulnerable atherosclerotic plaque, which is an unstable collection of
lipids (fatty acids) and white blood cells (especially macrophages) in the wall of an
artery. The resulting ischemia (restriction in blood supply) and oxygen shortage, if
left untreated for a sufficient period of time, can cause damage or death (infarction)
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of heart muscle tissue (myocardium). Heart attacks are the leading cause of death in
the United States for both men and women.
Heart arrhythmias, or abnormal activity (palpitations), occur when the
electrical impulses in the heart that coordinate heartbeats don't work properly,
causing the heart to beat too rapidly, too slowly or irregularly. Heart arrhythmias
are often harmless. Most people have occasional irregular heartbeats that may feel
like a fluttering or racing heart. However, some heart arrhythmias may cause
bothersome — sometimes even life-threatening — signs and symptoms. Treating
heart arrhythmia can often control or eliminate irregular heartbeats. In addition,
because troublesome heart arrhythmias are often made worse — or are even caused
— by a weak or damaged heart, one may be able to reduce one’s risk for arrhythmia
by adopting a heart-healthy lifestyle.
1% of live births display a congenital heart defect (CHD), which is a
malformation of the heart structure and major vessels that is present at birth. Many
types of heart defects exist, most of which either obstruct blood flow within the
heart or in vessels near it, or cause blood to flow through the heart in an abnormal
pattern. Heart defects are among the most common birth defects and are the leading
cause of birth defect-related deaths. Among individuals born with a congenital heart
defect many do not require treatment, however some complex CHDs necessitate
medication or surgery (Slide 2).
The circulatory system consists of the heart, blood cells and an intricate
system of blood vessels. It provides nourishment to organs (nutrients and oxygen)
and removes toxins and carbon dioxide from the peripheral organs. The circulatory
system is the first functional unit that forms in the developing embryo. The beating
heart can be visualized in the chick embryo as early as two days after fertilization; in
the mouse the heart starts beating by embryonic day e9.5. The heart is the main
organ that pumps blood through the blood vessels. In birds and mammals, the heart
contains four chambers, two atria and two ventricles, which are separated by the
atrioventricular valves (Slide 3).
There are three types of muscle in the body: 1) skeletal muscle, 2) smooth
muscle, and 3) heart muscle. Skeletal muscle is composed of elongated
multinucleate cells called myofibrils. Bundles of myofibrils are gathered together in
fascicles surrounded by a fibrous sheath. Myofibrils control voluntary movements of
the body and are innervated by motor neurons. Smooth muscle cells exist as
bundles of individual, spindle-shaped mononuclear cells. These cells contain the
same contractile apparatus as skeletal muscle, but it is not arranged in visible
sarcomeres (hence the designation “smooth”). Smooth muscle is found mainly
around the gut, blood vessels and ducts of glands, for which inherent rhythmic
contraction is required for function. Muscle movement in these organs is
involuntary. Lastly, cardiac muscle tissue is found only in the heart. Like skeletal
muscle, it has visible myofibrils. The cells are mostly mononuclear, but there is some
controversy regarding this issue (i.e. they may possess two nuclei). Heart muscle
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cells are joined by intercalating disks that contains structural junctions (adherens
junctions and gap junctions) to allow rapid spread of electrical signals through the
myocardium. Skeletal and cardiac muscle cells are post-mitotic and cannot divide.
However, they can grow via cell enlargement (Slide 4).
The three types of muscle cells (skeletal, smooth and cardiac) are all derived
from the mesoderm. However, during embryonic development the mesoderm
becomes specified into distinct types by the differential activity of Nodal signaling.
The paraxial mesoderm that surrounds the developing neural tube gives rise to
somites. During differentiation, the somites generate the myotome, which is the
precursor of skeletal muscles. Heart muscles cells, smooth muscle cells and the
circulatory system are derived from a different type of mesodermal tissue called the
lateral plate mesoderm. This tissue is found most distal to the developing neural
tube (Slide 5-6).
Formation of the heart from the splanchnic lateral plate mesoderm is well
understood for developing chick embryos. When the embryo is 18-20 hours old, the
presumptive heart cells move anteriorly between the ectoderm and endoderm,
toward the middle of the embryo, and remain in close contact with the endodermal
surface. They give rise to cell of the endocardial primordia (the endothelial cells
lining the inside of the heart, not to be confused with endoderm). While the foregut
arises by an inward folding of the splanchnopleure, this process also brings the
two cardiac tubes together. If this movement is disrupted, the embryo exhibits a
condition called cardia bifida (“two hearts”). Finally, the two chambers fuse together
to form the initial tube of endocardium plus myocardium, which will ultimately
give rise to the heart (Slide 7).
Cardiac morphogenesis in humans requires complex movements and
reorganization of presumptive heart tissue. On day 21, the heart is a singlechambered tube, and specification of the tube regions occurs progressively. During
this specification, the original tube undergoes looping, which places the
presumptive atria anterior to the presumptive ventricles. Next, the cushions of the
heart fuse together. Following this, the atrial and ventricular septa grow toward the
endocardial cushion by day 33, thereby separating the heart into four chambers.
The human heart is fully formed by three months, however there are still
perforations between atria that remain for a few additional months (Slide 8).
Several secreted factors are required to specify heart progenitors in the
developing embryo. Wnt proteins secreted from the neural tube are blocked by Wnt
inhibitors (Cerberus or Dkk) to specify the anterior lateral plate mesoderm. BMPs
and FGF8 further restrict this tissue to the cardiogenic (heart-forming) lineage.
Wnts promote specification of the posterior lateral plate mesoderm, which becomes
blood and blood vessels (Slide 9).
Differential gene expression refers to a cell turning on only the subset of
genes in its nucleus that are required for its function in the body. During
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development, many genes are expressed at early times and shut off later, while
others are only turned on in the final stages differentiation when the cell is mature.
One goal of stem cell research is to identify and control genes that allow a stem cell
to divide and maintain its pluripotency, as well as turning these genes off and
expressing different genes when we want cells to differentiate terminally, either in a
dish or in a patient to treat diseases. The BMP signaling pathway is an important
pathway for specification of heart tissue. The immature progenitor cells express
the BMP receptor type II (BMP-RII) on their surface. When these cells encounter
BMP signals in the developing embryo, they differentiate into heart cells.
Differentiation for the heart cell is characterized by the process of switching off a
large number of genes in the genome, except those genes that are specifically
needed in heart cells. One gene that is maintained is the Myosin light chain 2 gene,
which is present in all mature cardiomyocytes and encodes a component of the
molecular mechanism that allows these cells to contract (Slide 10).
The heart is composed of both muscle and non-muscle cell lineages. During
heart formation, differentiation of progenitor cells into these multiple lineages is
under tight spatial and temporal control. Mesodermal precursors are marked by
expression of the transcription factor Brachyury (Bry). As the mesodermal
precursors begin to acquire cardiogenic potential (i.e. the ability to become heartproducing cells), they begin to express two important transcription factors (i.e. DNA
binding proteins): Nkx2.5 and Isl1. Progenitor cells that will give rise to the left
ventricle are called the 1st heart field progenitor cells and are partitioned early in
development. They continue to express the transcription factor Nkx2.5 but turn off
Isl-1 and instead express the cell surface protein c-Kit. Little more is known about
progenitor cells of the 1st heart field and their derivatives, which give rise to most of
the left ventricular chamber. Cells within the 2nd heart field are multipotent, Isl-1+
cardiovascular progenitors. They give rise to all three major cell lineages of the
heart: cardiomyocytes, smooth muscle cells (SMCs) and to a limited extent
endothelial cells (ECs). The multipotent cardiac progenitors of the 2nd heart field
produce cells of the right ventricle, outflow tract and proximal coronary arteries.
They express the transcription factors Nkx2.5 and GATA4. Vascular progenitors
eventually lose expression of Nkx2.5 and GATA4 as they acquire the ability to
produce blood vessels (Slide 11).
Differentiation of cardiac progenitors into cells that will give rise to the atria,
right ventricle and smooth muscle cells of the heart outflow tract occurs through a
secondary lineage specification of progenitors, followed by the process of
differentiation. The best markers for differentiated heart muscle cells (regardless of
their eventual atrial or ventricular fate) is cardiac troponin T (cTnT) and myosin
light chain 2 (MLC2). MLC2a and MLC2v are two distinct isoforms of the myosin
light chain regulatory protein subunit that are expressed specifically in the atria or
ventricles, respectively. These genes are usually turned OFF in the immature
progenitor cells, but are turned ON as the progenitors differentiate and form mature
atrial or ventricular cardiomyocytes. The heart also contains nodal (pacemaker)
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cells that coordinate rhythmic beating of the heart tissue. The best marker for nodal
(pacemaker) cells is Hcn4, an ion channel found in the cell membrane (Slide 12).
One source for transplantation-based replacement therapies of
cardiomyocytes is directed differentiation in vitro of human ES cells into
cardiomyocytes, using defined growth factors. A mural graft is one that occurs in the
wall of either a blood vessel or chamber within the heart (from Latin murus = wall).
Three studies by: 1) Beltrami, A. P. et al. Cell 114, 763−776 (2003); 2) Oh, H. et al.
Proc. Natl Acad. Sci. USA 100, 12313−12381 (2003); and 3) Messina, E. et al. Circ.
Res. 95, 911−921 (2004) have independently identified other primitive cells from
the adult heart that are capable of dividing and developing into mature heart and
vascular cells. These cardiac stem cells are distinct from cardiac progenitors. Both
cell populations divide and renew themselves, however progenitor (Isl1+) cells are
committed to becoming heart cells whereas stem cells have the potential to form
many different cell types.
Two of these studies found stem cells in the heart of adult rats (1,2). These
cells do not express Isl1, but were isolated based on their expression of cell-surface
proteins (either c-kit or Sca-1) that are usually associated with stem cells derived
from bone marrow. In the first study, c-kit+ cells from rat heart were shown to be
self-renewing and capable of forming heart muscle cells and certain vascular cells.
Although these heart muscle cells fail to contract spontaneously in culture, they
seem able to regenerate functional heart muscle when injected into a damaged
heart. Therefore, it is not clear whether they represent bona fide cardiac stem cells.
In the second study, Sca-1+; c-kit- cells from mouse heart developed into heart
muscle when provided intravenously after injury, although this was in part due to
fusion with the heart cells of the host. It is to date unclear whether there are true
stem cells present in the heart. However, such cells do not represent a promising
source for generating cardiomyocytes (Slide 13-14).
Directed differentiation of mouse ES cells into cardiomyocytes is achieved by
removing leukemia inhibitory factor (LIF), which maintains cells in the
pluripotent state, and adding high concentrations of serum, which contains BMPs. In
the presence of high serum concentrations, the embryoid bodies which appear on
day 2 of the in vitro differentiation protocol will begin to differentiate toward the
mesodermal and cardiac lineage. Nkx2.5+ cardiomyogenic progenitors appear at
between 5-7 days in the differentiation media, whereas beating cardiomyocytes
appear after 7-10 days (Slide 15).
Directed differentiation of mouse ES cells into cardiomyocytes is relatively
inefficient. One can determine the efficiency of generating cardiac progenitors by
monitoring the frequency of Nkx2.5+ cardiac progenitors. This can be achieved by
either: a) staining for the Nkx2.5 transcription factor after 5 days in culture; or b)
using ES cells that are engineered to express eGFP under control of Nkx2.5, such
that when ES cells differentiate into cardiac progenitors they turn on eGFP. One can
then sort (separate) GFP-positive from GFP-negative cells and determine the
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frequency of these cells in the culture. Using this method, the authors found that
3.8% of cells become cardiac progenitors.
Why is the directed differentiation of ES cells into cardiomyocytes so
inefficient? Multipotent cardiovascular progenitors of the 2nd heart field produce
cells of the right ventricle, outflow tract and proximal coronary arteries and express
the transcription factors Nkx2.5 and GATA4. In addition, these cells also give rise to
vascular progenitors and will lose expression of Nkx2.5 and GATA4 as they acquire
the ability to produce blood vessels. These vascular progenitors, however, will
maintain expression of Isl1 and also express the cell surface receptor Flk-1 (or
VEGFR-1). During the directed differentiation of ES cells into cardiomyocytes, it
turns out that a large fraction of progenitors become Flk-1+ vascular progenitors
that can be isolated using FACS (fluorescence-activated cell sorting) via their ability
to express the Flk-1 receptor on their cell surface (Slide 16).
Why do heart cells that are generated in vitro beat spontaneously? This
is primarily a property of only a subset of heart cells called node cells. These cells
express a variety of proteins that are important for their beating properties, such as:
a) Cardiac troponin T - Troponin is a complex of three regulatory proteins that is
integral to contraction of skeletal and cardiac muscle but not smooth muscle.
Troponin is attached to the protein tropomyosin and lies within the groove
between actin filaments in muscle tissue. In relaxed muscle, tropomyosin blocks the
attachment site for the myosin cross-bridge, thus preventing contraction. When the
muscle cell is stimulated by an action potential, calcium channels open and release
calcium into the myoplasm. Some of this calcium attaches to troponin, which causes
it to change shape and exposes binding sites for myosin (active sites) on actin
filaments. Myosin binding to actin forms cross-bridges and contraction of the
muscle begins. Troponin is found in both skeletal muscle and cardiac muscle, but the
specific isoform of troponin differs between types of muscle. The main difference is
that the TnC subunit in skeletal muscle has four Ca++ binding sites, whereas in
cardiac muscle this subunit only has three.
b) HCN4 - The HCN4 gene encodes the pore-forming subunit of a hyperpolarizationactivated, cyclic nucleotide-modulated cation channel. HCN4 channels are the
predominant HCN isoform in the sinoatrial node and contribute to the pacemaker
current that controls rhythmic activity in the heart and brain.
c) Cav3.2: an important voltage-dependent Ca++ channel that regulates influx of Ca++
inside the cell, which stimulates muscle contraction (Slide 17).
Knowledge of signaling molecules that specify the identity of cardiomyoctes
has also been used to direct differentiation of human ES cells in vitro into heart
muscle cells. Directed differentiation of hES cells into cardiomyocytes requires high
amounts of FBS (fetal bovine serum). BMP4 can enhance the efficiency of producing
cardiomyocytes from human ES cells. However, it is only able to do this during the
first few days of treatment. The experimental protocol for directed differentiation of
human ES cells into cardiomyocyte takes a few additional days as compared to that
for mouse cells (Slide 18).
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Perhaps the most important application for human stem cells is generation of
cells and tissues that could be used for cell-based therapies. Today, donated organs
and tissues are often used to replace ailing or destroyed tissue, but the need for
transplantable tissues and organs far outweighs the available supply. Stem cells,
directed to differentiate into specific cell types, offer the possibility for a renewable
source of replacement cells and tissues to treat diseases, including many heart
diseases. It may become possible to generate healthy heart muscle cells in the
laboratory and then transplant those cells into patients with chronic heart diseases.
A number of stem cell types, including embryonic stem (ES) cells, cardiac
stem cells that naturally reside within the heart, myoblasts (muscle stem cells),
adult bone marrow-derived cells including mesenchymal cells (these bone
marrow-derived cells give rise to tissues such as muscle, bone, tendons, ligaments
and adipose tissue), endothelial progenitor cells (these give rise to the
endothelium, or the interior lining of blood vessels) and umbilical cord blood cells
have been investigated as possible sources for regenerating damaged heart tissue.
All have been explored in mouse or rat models, and some have been tested in larger
animal models, such as pigs. The best model to investigate the contributions of
various stem cells to heart regeneration is to induce a heart infarction by ligating
(tying off) the coronary artery, which supplies oxygen to the heart itself (Slide 19).
Preliminary research in mice and other animals indicates that bone marrow
stromal cells, when transplanted into a damaged heart, can have beneficial effects.
Whether these cells can generate heart muscle cells, stimulate the growth of new
blood vessels that repopulate the heart tissue or assist via some other mechanism is
actively under investigation. For example, injected cells may accomplish repair by
secreting growth factors rather than actually incorporating into the heart. Promising
results from animal studies have served as the basis for a small number of
exploratory studies in humans (Slide 20).
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