0. Directed differentiation of ES cells into cardiac mesoderm

1. Directed differentiation allows production of a desired cell type
Pluripotent
Multipotent
Differentiated
cells
Heart muscle cells (or cardiomyocytes) derived by this process can be used for:
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.
Ectodermal cell
brain
ES cell
• ES cells from the inner cell mass can give rise to all three germ layers and are pluripotent.
• Cells in each germ layer retain the ability to proliferate and 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); 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.
Mesodermal cell
heart
pancreas
Endodermal cell 1 1

2. Diseases of heart muscle tissue
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 large number of research laboratories have focused their efforts in developing methods to direct differentiation of ES cells into cardiomyocytes.
What are cardiomyocytes? Cardiomyocytes are the primary beating cells in the heart. These cells are coupled by junctions, including gap junctions that allow the signals to spread very rapidly from one cell to the other.
Why do we care?
Cardiovascular disease (CVD), which includes hypertension, coronary heart disease, stroke, and congestive heart failure, has ranked as the number one cause of death in the United States every year since 1900 except 1918, when the nation struggled with an influenza epidemic. Nearly 2600 Americans die of CVD each day, roughly one person every 34 seconds. Given the aging of the population and the 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, the overstretching of viable cardiac cells attempting to sustain cardiac output, leading to heart failure, and eventual death. Restoring damaged heart muscle tissue, through repair or regeneration, is therefore a potentially new strategy 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, causing heart cells to die. This is most commonly due to occlusion (blockage) of a coronary artery following the 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) 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) Heart rhythm problems (heart arrhythmias) occur when the electrical impulses in your heart that coordinate your heartbeats don't work properly, causing your heart to beat too fast, too slow 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. Heart arrhythmia treatment 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, you may be able to reduce your arrhythmia risk by adopting a heart-healthy lifestyle.
1% of live births display a congenital heart defect. A congenital heart defect (CHD) is a defect in the structure of the heart and great vessels which is present at birth. Many types of heart defects exist, most of which either obstruct blood flow in the heart or 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. Approximately 9 people in 1000 are born with a congenital heart defect. Many defects don't need treatment, but some complex congenital heart defects require medication or surgery. 2

3. A four-chambered heart underlies double circulation (birds & mammals)
The circulatory system is:
Heart
Blood vessels
Blood cells
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 & amphibians).
Right atrium
Left atrium
Atrioventricular valves
• The circulatory system consists of the heart, the 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.
Right ventricle
Left ventricle 3 3

4. Heart muscle, to perform its unique function, is different from skeletal and smooth muscle
1. Cardiac muscle cells:
Have visible myofibrils
Occur only in heart
Involuntary movement
Cells joined by intercalating disks to allow rapid spread of electrical signals
2. Skeletal muscle cells:
Elongated multinucleate cells called myofibrils.
Voluntary movements
3. Smooth muscle cells:
Individual, spindle-shaped mononuclear cells
Found in gut, blood vessels and ducts of glands
Involuntary movements
Three types of muscle cells
• There are three types of muscle in the body: 1) skeletal muscle, 2) smooth muscle, and 3) heart muscle
The 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 the skeletal muscle, but this is not arranged in visible sarcomeres (hence the designation smooth). Smooth muscle is found mainly around the gut, blood vessels and ducts of glands in which inherent rhythmic contraction is required for function. Muscle movement in these organs is involuntary.
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. may be binucleate). The cells are joined by intercalating disks that contains structural junctions (adherens junctions and gap junctions) to allow the 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. 4 4

5. How are cardiomyocytes generated during development?
Lineage restrictions
Pluripotent
Multipotent
Differentiation
Ectodermal cell ? ?
What are the signals that regulate the differentiation of heart cells (cardiomyocytes) from undifferentiated progenitors?
ES cell
Mesodermal cell
Multipotent
cardiovascular
progenitor
Cardiomyocyte
Endodermal cell 5

6. Heart, blood and blood vessels are derived from the lateral mesoderm
The three types of muscle cells (skeletal, smooth and cardiac) are all derived from the mesoderm.
However during embryonic development, the mesoderm gets 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.
The 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.
Heart, circulatory system, smooth muscle
Skeletal muscles 6 6

7. Formation of heart tube from germ layers (in the chick)
• The above diagram demonstrates the formation of the heart from the splanchnic lateral plate mesoderm in developing chick embryos.
When the embryo is 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).
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 the endocardium plus myocardium, which will give rise to the heart. 7 7

8. Cardiac looping and chamber formation in the human embryo
A schematic diagram of cardiac morphogenesis in humans. On day 21, the heart is a single-chambered tube.
Specification of the tube regions occurs progressively.
During specification of these regions, the original tube undergoes looping, placing the presumptive atria anterior to the presumptive ventricles.
The cushions of the heart fuse together
The atrial and ventricular septa grow toward the endocardial cushion by day 33, thus separating the heart into four chambers.
The heart is fully formed by three months. However there are still perforations between atria that remain for a few additional months. 8 8

9. Secreted factors specify heart-forming mesoderm in vivo
Lineage restrictions
Differentiation
FGF8
Cardiogenic
mesodermal
cell
Anterior lateral plate mesodermal cell
Cardiomyocyte
Cerberus
Wnts
BMPs
Lateral plate mesodermal cell
Wnts secreted from the neural tube are blocked by the 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.
Noggin
Posterior lateral plate mesodermal cell
Hemangiogenic
mesodermal cell
Blood cells 9

10. Distinct cell signaling pathways induce cardiomyocyte-specific genes
Gene expression
Heart muscle cell
(Cardiomyocyte)
BMPs
BMP-RII
Progenitor
cell
OFF
• 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 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 the specification of the 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 the heart cells.
The process of differentiation of the heart cell is characterized by the process of switching off a large number of genes in the genome, except the genes that are specifically expressed in these cells. One of the genes that is maintained is the Myosin light chain 2 gene which is present in all mature cardiomyocytes.
OFF ON
Heart muscle cell
(Cardiomyocyte)
Myosin light chain 2 gene 10 10

11. Markers of the cardiomyocyte progenitor lineage
Cardiomyocyte lineage
Differentiation
Bry
Nkx2.5+ / Isl-1+
Nkx2.5+ / c-Kit+
1st heart field
progenitor
Left ventricle
Cardiogenic
mesodermal
progenitor
Mesodermal
Precursor
Nkx2.5+ / Isl-1+/ GATA4+
Cardiac
progenitor
Remainder of heart
The heart is composed of muscle and non-muscle cell lineages.
During heart formation, differentiation of progenitor cells into these multiple heart cell lineages is under tight spatial and temporal control.
Mesodermal precursors are marked by expression of the transcription factor Bry.
As the mesodermal precursors begin to acquire cardiogenic potential (i.e. the ability to become heart-producing 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. These are partitioned early in development. They continue to express the transcription factor Nkx2.5 but not Isl-1. Instead, they express the cell surface protein c-Kit. However, little 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 in 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 lose expression of Nkx2.5 and GATA4 as they acquire the ability to produce blood vessels.
Nkx2.5+ / Isl-1+/ GATA4+/ Flk-1+
Isl-1+/ Flk-1+
Multipotent cardiovascular
progenitor
(2nd heart field)
Blood vessels
and blood cells
Vascular
progenitor 11

12. Markers of the mature cardiomyocyte lineage
Differentiation of cardiac progenitors into cells that will give rise to the atria, right ventricle and smooth muscle cells of the outflow tract in the heart 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 they are turned ON as the progenitors differentiate and form mature atrial or ventricular cardiomyocytes.
The heart also contains nodal (pacemaker) cells. These cells coordinate the rhythmic beating of the heart tissue. The best marker for nodal (pacemaker) cells is Hcn4, an ion channel found in the cell membrane. 12

13. Sources of cells for cardiomyocyte replacement therapies: the dish
Directed
differentiation
Transplantation &
engraftment
Stable formation
of mural grafts
One of the sources 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).

14. Sources of cells for cardiomyocyte replacement therapies: the heart
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.
The progenitor cells (Isl1+ cells) are committed to becoming heart cells.
The stem cells have the potential to form several 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 the presence 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 the 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 were able to develop 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.

15. Directed differentiation protocol for mouse ES cells into cardiomyocytes
mES cells
- LIF
+ serum
Day 2
EBs
Day 5-7
Nkx2.5+
progenitors
Day 7-10
beating
cardiomyocytes
Wnt/BMP/Activin and serum are often used to induce cardiomyocyte precursors
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 concentration of serum, the embryonic bodies which appear at day 2 of the in vitro differentiation protocol will begin to differentiate toward the mesodermal and cardiac lineage.
The Nkx2.5+ cardiomyogenic progenitors appear between 5-7 days in the differentiation media, whereas beating cardiomyocytes appear between 7-10 days.
2 days
4 days
2 - 4 days
ES cells
EBs
Nkx2.5+
cardiomyogenic
progenitors
Beating
cardiomyocytes 15 15

16. Directed differentiation of mouse ES cells produces vascular and cardiac lineages
Isl-1+/ Flk-1+
Nkx2.5+ / Isl-1+/ Gata4+
Vascular
progenitors
Cardiac
progenitors
Fraction of Flk-1+
progenitors at day 3-5
Fraction of Nkx2.5+
progenitors at day 5 % %
The process of the directed differentiation of the mouse ES cells into the cardiomyocytes is relatively inefficient. One can determine the efficiency of the generation of cardiac progenitors by monitoring the frequency of Nkx2.5+ cardiac progenitors.
This can be achieved either by several methods: a) staining for Nkx2.5 transcription factor after 5 days in culture; b) use ES cells that are engineered to express eGFP into the Nkx2.5 gene such that when ES cells are differentiated into cardiac progenitors they turn on eGFP. Then one can sort (separate) GFP positive from GFP negative cells and determine the frequency of these cells in the culture. Using this method, the authors have found that 3.84% of cells become cardiac progenitors.
Why is the directed differentiation of ES cells into cardiomyocytes so inefficient?
The multipotent cardiovascular progenitors in 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. 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 maintain expression of Isl1 and also express the cell surface receptor Flk-1 (or VEGFR-1).
During the process of directed differentiation of ES cells into cardiomyocytes, a large fraction of progenitors become Flk-1-positive vascular progenitors that can be isolated by FACS via their ability to express the Flk-1 receptor on their cell surface. %
3.84 %
Nkx2.5::eGFP 16 16

17. Isolated heart muscle cells beat spontaneously
Directed differentiation of ES cells creates specialized beating heart cells in vitro.
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.
• Why do heart cells that are generated in vitro beat spontaneously?
This is mostly a property of only a subset of heart cells called the node cells. These cells express a variety of proteins that are important for beating properties such as:
Cardiac troponin T: Troponin is a complex of three regulatory proteins that is integral to muscle contraction in 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 a relaxed muscle, tropomyosin blocks the attachment site for the myosin cross-bridge, thus preventing contraction. When the muscle cell is stimulated to contract by an action potential, calcium channels open in the myoplasmic membrane and release calcium into the myoplasm. Some of this calcium attaches to troponin which causes it to change shape, exposing binding sites for myosin (active sites) on the actin filaments. Myosin binding to actin forms cross bridges and contraction (cross bridge cycling) of the muscle begins. Troponin is found in both skeletal muscle and cardiac muscle, but the specific versions of troponin differ between types of muscle. The main difference is that the TnC subunit of troponin in skeletal muscle has four calcium ion binding sites, whereas in cardiac muscle there are only three. The actual amount of calcium that binds to troponin varies widely.
b) HCN4 - The HCN4 gene encodes the pore-forming subunit of a hyperpolarization-activated, cyclic nucleotide-modulated cation channel. HCN4 channels are the predominant HCN isoform in the sinoatrial node and contribute to pacemaker current that controls rhythmic activity in the heart and brain.
c) Cav3.2: is an important voltage-dependent Ca++ channel that regulates the influx of Ca++ inside the cell which stimulates muscle contractions. 17 17

18. Directed differentiation protocol for human ES cells to cardiomyocytes
- bFGF
+ 20% FBS
- bFGF
+ 20% FBS
- bFGF
+ 20% FBS
BMP-4
Day 7
Nkx2.5+
progenitors
Day 11
beating
cardiomyocytes
hES cells
Day 4
hEBs
Wnt/BMP/Activin and serum are often used to induce cardiomyocyte precursors
Identification of signaling molecules that specify the identity of cardiomyoctes has also been used to direct differentiation of human ES cells into heart muscle cells in vitro.
Directed differentiation of human ES cells into cardiomyocytes requires high amounts of FBS (fetal bovine serum). BMP4 can enhance the efficiency of generating cardiomyocytes from human ES cells. However, it is only able to do this in the first few days of treatment.
The process of directed differentiation of human ES cells into cardiomyocyte takes a few additional days, compared to the mouse.
4 days
3 days
4 days
20% FBS
BMP-4
ng/ml
20% FBS
20% FBS
hES cells
Human
embryoid
bodies
Nkx2.5+
cardiomyogenic
progenitors
Beating
cardiomyocytes 18 18

19. Rodent model of heart attack to test stem cell-based repair of heart tissue
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
Rats, mice and pigs are commonly used to test whether cells can repair hearts
Conditions mimicking those of a heart attack are achieved in animal models by ligation of the coronary artery
Perhaps the most important potential application of human stem cells is providing cells and tissues that can 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 of 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 them into patients with chronic heart disease.
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 normally give rise to 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 sources for regenerating damaged heart tissue. All have been explored in mouse or rat models, and some have been tested in larger animals such as pigs.
The best model to investigate the contribution of various stem cells to heart regeneration is to induce a myocardial infarction (heart attack) by tying off (ligating) the coronary artery.

20. Stem cells directly or indirectly repair damaged heart tissue
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 in fact 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 stimulate 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. 20 20

21. Summary
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. 21 21