Archive for the ‘Cardiac Stem Cells’ Category
Stem cells: What they are and what they do Stem cells and derived products offer great promise for new medical treatments. Learn about stem cell types, current and possible uses, ethical issues, and the state of research and practice. By Mayo Clinic Staff
You’ve heard about stem cells in the news, and perhaps you’ve wondered if they might help you or a loved one with a serious disease. You may wonder what stem cells are, how they’re being used to treat disease and injury, and why they’re the subject of such vigorous debate.
Here are some answers to frequently asked questions about stem cells.
Researchers and doctors hope stem cell studies can help to:
Generate healthy cells to replace diseased cells (regenerative medicine). Stem cells can be guided into becoming specific cells that can be used to regenerate and repair diseased or damaged tissues in people.
People who might benefit from stem cell therapies include those with spinal cord injuries, type 1 diabetes, Parkinson’s disease, Alzheimer’s disease, heart disease, stroke, burns, cancer and osteoarthritis.
Stem cells may have the potential to be grown to become new tissue for use in transplant and regenerative medicine. Researchers continue to advance the knowledge on stem cells and their applications in transplant and regenerative medicine.
Test new drugs for safety and effectiveness. Before using new drugs in people, some types of stem cells are useful to test the safety and quality of investigational drugs. This type of testing will most likely first have a direct impact on drug development for cardiac toxicity testing.
New areas of study include the effectiveness of using human stem cells that have been programmed into tissue-specific cells to test new drugs. For testing of new drugs to be accurate, the cells must be programmed to acquire properties of the type of cells to be tested. Techniques to program cells into specific cells continue to be studied.
For instance, nerve cells could be generated to test a new drug for a nerve disease. Tests could show whether the new drug had any effect on the cells and whether the cells were harmed.
Stem cells are the body’s raw materials cells from which all other cells with specialized functions are generated. Under the right conditions in the body or a laboratory, stem cells divide to form more cells called daughter cells.
These daughter cells either become new stem cells (self-renewal) or become specialized cells (differentiation) with a more specific function, such as blood cells, brain cells, heart muscle or bone. No other cell in the body has the natural ability to generate new cell types.
Researchers have discovered several sources of stem cells:
Embryonic stem cells. These stem cells come from embryos that are three to five days old. At this stage, an embryo is called a blastocyst and has about 150 cells.
These are pluripotent (ploo-RIP-uh-tunt) stem cells, meaning they can divide into more stem cells or can become any type of cell in the body. This versatility allows embryonic stem cells to be used to regenerate or repair diseased tissue and organs, although their use in people has been to date limited to eye-related disorders such as macular degeneration.
Adult stem cells. These stem cells are found in small numbers in most adult tissues, such as bone marrow or fat. Compared with embryonic stem cells, adult stem cells have a more limited ability to give rise to various cells of the body.
Until recently, researchers thought adult stem cells could create only similar types of cells. For instance, researchers thought that stem cells residing in the bone marrow could give rise only to blood cells.
However, emerging evidence suggests that adult stem cells may be able to create unrelated types of cells. For instance, bone marrow stem cells may be able to create bone or heart muscle cells. This research has led to early-stage clinical trials to test usefulness and safety in people. For example, adult stem cells are currently being tested in people with neurological or heart disease.
This new technique may allow researchers to use these reprogrammed cells instead of embryonic stem cells and prevent immune system rejection of the new stem cells. However, scientists don’t yet know if altering adult cells will cause adverse effects in humans.
Researchers have been able to take regular connective tissue cells and reprogram them to become functional heart cells. In studies, animals with heart failure that were injected with new heart cells experienced improved heart function and survival time.
Perinatal stem cells. Researchers have discovered stem cells in amniotic fluid in addition to umbilical cord blood stem cells. These stem cells also have the ability to change into specialized cells.
Amniotic fluid fills the sac that surrounds and protects a developing fetus in the uterus. Researchers have identified stem cells in samples of amniotic fluid drawn from pregnant women during a procedure called amniocentesis, a test conducted to test for abnormalities.
More study of amniotic fluid stem cells is needed to understand their potential.
Embryonic stem cells are obtained from early-stage embryos a group of cells that forms when a woman’s egg is fertilized with a man’s sperm in an in vitro fertilization clinic. Because human embryonic stem cells are extracted from human embryos, several questions and issues have been raised about the ethics of embryonic stem cell research.
The National Institutes of Health created guidelines for human stem cell research in 2009. Guidelines included defining embryonic stem cells and how they may be used in research and donation guidelines for embryonic stem cells. Also, guidelines stated embryonic stem cells may only be used from embryos created by in vitro fertilization when the embryo is no longer needed.
The embryos being used in embryonic stem cell research come from eggs that were fertilized at in vitro fertilization clinics but never implanted in a woman’s uterus. The stem cells are donated with informed consent from donors. The stem cells can live and grow in special solutions in test tubes or petri dishes in laboratories.
Although research into adult stem cells is promising, adult stem cells may not be as versatile and durable as are embryonic stem cells. Adult stem cells may not be able to be manipulated to produce all cell types, which limits how adult stem cells can be used to treat diseases.
Adult stem cells also are more likely to contain abnormalities due to environmental hazards, such as toxins, or from errors acquired by the cells during replication. However, researchers have found that adult stem cells are more adaptable than was initially suspected.
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Stem cells: What they are and what they do – Mayo Clinic
Heart transplants have been around since 1967, but they’re still anything but routine. In an effort to ensure a steady supply of compatible organs, a team of scientists from Massachusetts General Hospital (MGH) is working on ways to create bioengineered human hearts by first stripping donor hearts of cells that could provoke an immune response in a potential recipient, and then using the recipient’s own induced pluripotent stem cells (iPSCs) to generate cardiac muscle cells that can be used to repopulate the heart in an automated bioreactor system.
Every year, 800,000 people worldwide have heart conditions that require a transplant. Unfortunately, there are only enough suitable donor hearts for around 3,500 operations. Part of the reason for this isn’t that there aren’t enough healthy hearts donated to go around, but that a heart needs to be biologically compatible with the recipient.
And even if there is an extremely close tissue match, the recipient’s body will treat still the new heart as alien and attack it. To prevent this tissue rejection, the recipient’s autoimmune system must be suppressed by a battery of pills for a lifetime, combined with another battery of pills to correct the damage caused by suppressing the immune system.
What the MGH team led by Dr Harald Ott is trying to achieve is a way of turning the alien heart into a not-alien one. In other words, to make it as much like the recipient’s original heart from a cellular point of view. This way, the body is less likely to reject it and the follow-up medical regime can be less aggressive.
The MGH approach to essentially take a donor heart, strip it down and rebuild it much as one might strip a house down to its frame and then rebuild it with all-new materials. The heart, like most organs, consists of living cells that are held in place by a connective matrix made of collagen fibers. It’s the living cells that allow the heart to pump blood, but they’re also what spark an immune reaction in the host body, so the idea is to remove the original cells, then replace them in the remaining collagen matrix with cells created from the recipient’s own. Since the new cells are genetically identical to those of the recipient, tissue rejection is less likely.
According to MGH, Dr Ott had already developed a procedure in 2008 that allowed him to remove living cells from organs using a detergent solution. The MGH team then used the leftover extracellular matrix as a scaffold that can then be repopulated with new cells. In this way, they could not only create working rat lungs and kidneys, but also decellularized large-animal hearts, lungs, and kidneys.
The next step was to scale up the method on a whole human heart. This was done by creating iPSCs. The iPSCs are made by using a new method to reprogram skin cells with messenger RNA factors so they revert to an embryonic state.
These all-purpose stem cells can then be induced to become any kind of cell in the human body. In this case, they were turned into cardiac muscle cells, or cardiomyocytes. According to MGH, this method not only is more efficient and allows for creating cells in large enough quantities for clinical use, but it also avoids many regulatory obstacles that more conventional methods come up against.
These cardiac cells were then introduced into 73 decellularized human hearts from donors who were brain dead or had suffered cardiac death. The hearts selected weren’t suitable for transplant, so were used with consent for research purposes. The cells were reseeded into the 3D matrix of the left ventricular wall of the decellularized hearts as thin slices, then as 15 mm fibers, which began to contract on their own within days.
The hearts were then placed for 14 days in an automated bioreactor system developed by the MGH team. This provided the tissues with nourishment in the form of a solution while ventricular pressure and other stressors were applied to exercise them. The researchers say the result was dense regions of iPSC-derived cells that resembled immature cardiac muscle tissue and contracted like heart tissue when subjected to electrical stimulation.
“Regenerating a whole heart is most certainly a long-term goal that is several years away, so we are currently working on engineering a functional myocardial patch that could replace cardiac tissue damaged due to a heart attack or heart failure,” says Jacques Guyette, PhD, of the MGH Center for Regenerative Medicine (CRM). “Among the next steps that we are pursuing are improving methods to generate even more cardiac cells recellularizing a whole heart would take tens of billions optimizing bioreactor-based culture techniques to improve the maturation and function of engineered cardiac tissue, and electronically integrating regenerated tissue to function within the recipient’s heart.”
The team’s results were was published in Circulation Research.
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Stripping donor hearts and repopulating them with …
Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells, capable of germline transmission, from mouse somatic cells by transduction of four defined transcription factors. Here, we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. Furthermore, these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts.
Embryonic stem (ES) cells, derived from the inner cell mass of mammalian blastocysts, have the ability to grow indefinitely while maintaining pluripotency (Evans and Kaufman, 1981andMartin, 1981). These properties have led to expectations that human ES cells might be useful to understand disease mechanisms, to screen effective and safe drugs, and to treat patients of various diseases and injuries, such as juvenile diabetes and spinal cord injury (Thomson etal., 1998). Use of human embryos, however, faces ethical controversies that hinder the applications of human ES cells. In addition, it is difficult to generate patient- or disease-specific ES cells, which are required for their effective application. One way to circumvent these issues is to induce pluripotent status in somatic cells by direct reprogramming (Yamanaka, 2007).
We showed that induced pluripotent stem (iPS) cells can be generated from mouse embryonic fibroblasts (MEF) and adult mouse tail-tip fibroblasts by the retrovirus-mediated transfection of four transcription factors, namely Oct3/4, Sox2, c-Myc, and Klf4 (Takahashi and Yamanaka, 2006). Mouse iPS cells are indistinguishable from ES cells in morphology, proliferation, gene expression, and teratoma formation. Furthermore, when transplanted into blastocysts, mouse iPS cells can give rise to adult chimeras, which are competent for germline transmission (Maherali etal., 2007, Okita etal., 2007andWernig etal., 2007). These results are proof of principle that pluripotent stem cells can be generated from somatic cells by the combination of a small number of factors.
In the current study, we sought to generate iPS cells from adult human somatic cells by optimizing retroviral transduction in human fibroblasts and subsequent culture conditions. These efforts have enabled us to generate iPS cells from adult human dermal fibroblasts and other human somatic cells, which are comparable to human ES cells in their differentiation potential in vitro and in teratomas.
Induction of iPS cells from mouse fibroblasts requires retroviruses with high transduction efficiencies (Takahashi and Yamanaka, 2006). We, therefore, optimized transduction methods in adult human dermal fibroblasts (HDF). We first introduced green fluorescent protein (GFP) into adult HDF with amphotropic retrovirus produced in PLAT-A packaging cells. As a control, we introduced GFP to mouse embryonic fibroblasts (MEF) with ecotropic retrovirus produced in PLAT-E packaging cells(Morita etal., 2000). In MEF, more than 80% of cells expressed GFP (FigureS1). In contrast, less than 20% of HDF expressed GFP with significantly lower intensity than in MEF. To improve the transduction efficiency, we introduced the mouse receptor for retroviruses, Slc7a1 (Verrey etal., 2004) (also known as mCAT1), into HDF with lentivirus. We then introduced GFP to HDF-Slc7a1 with ecotropic retrovirus. This strategy yielded a transduction efficiency of 60%, with a similar intensity to that in MEF.
The protocol for human iPS cell induction is summarized inFigure1A. We introduced the retroviruses containing human Oct3/4, Sox2, Klf4, and c-Myc into HDF-Slc7a1 (Figure1B; 8 105 cells per 100 mm dish). The HDF derived from facial dermis of 36-year-old Caucasian female. Six days after transduction, the cells were harvested by trypsinization and plated onto mitomycin C-treated SNL feeder cells (McMahon and Bradley, 1990) at 5 104 or 5 105 cells per 100 mm dish. The next day, the medium (DMEM containing 10% FBS) was replaced with a medium for primate ES cell culture supplemented with 4 ng/ml basic fibroblast growth factor (bFGF).
Induction of iPS Cells from Adult HDF
(A) Time schedule of iPS cell generation.
(B) Morphology of HDF.
(C) Typical image of non-ES cell-like colony.
(D) Typical image of hES cell-like colony.
(E) Morphology of established iPS cell line at passage number 6 (clone 201B7).
(F) Image of iPS cells with high magnification.
(G) Spontaneously differentiated cells in the center part of human iPS cell colonies.
(HN) Immunocytochemistry for SSEA-1 (H), SSEA-3 (I), SSEA-4 (J), TRA-1-60 (K), TRA-1-81 (L), TRA-2-49/6E (M), and Nanog (N). Nuclei were stained with Hoechst 33342 (blue). Bars = 200 m (BE, G), 20 m (F), and 100 m (HN).
Approximately two weeks later, some granulated colonies appeared that were not similar to hES cells in morphology (Figure1C). Around day 25, we observed distinct types of colonies that were flat and resembled hES cell colonies (Figure1D). From 5 104 fibroblasts, we observed 10 hES cell-like colonies and 100 granulated colonies (7/122, 8/84, 8/171, 5/73, 6/122, and 11/213 in six independent experiments, summarized in Table S1). At day 30, we picked hES cell-like colonies and mechanically disaggregated them into small clumps without enzymatic digestion. When starting with 5 105 fibroblasts, the dish was nearly covered with more than 300 granulated colonies. We occasionally observed some hES cell-like colonies in between the granulated cells, but it was difficult to isolate hES cell-like colonies because of the high density of granulated cells. The nature of the non-hES-like cells remains to be determined.
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Induction of Pluripotent Stem Cells from Adult Human …
The heart is a muscular organ in humans and other animals, which pumps blood through the blood vessels of the circulatory system. Blood provides the body with oxygen and nutrients, and also assists in the removal of metabolic wastes. The heart is located in the middle compartment of the mediastinum in the chest.
In humans, other mammals, and birds, the heart is divided into four chambers: upper left and right atria; and lower left and right ventricles. Commonly the right atrium and ventricle are referred together as the right heart and their left counterparts as the left heart. Fish in contrast have two chambers, an atrium and a ventricle, while reptiles have three chambers. In a healthy heart blood flows one way through the heart due to heart valves, which prevent backflow. The heart is enclosed in a protective sac, the pericardium, which also contains a small amount of fluid. The wall of the heart is made up of three layers: epicardium, myocardium, and endocardium.
The heart pumps blood through the body. Blood low in oxygen from the systemic circulation enters the right atrium from the superior and inferior venae cavae and passes to the right ventricle. From here it is pumped into the pulmonary circulation, through the lungs where it receives oxygen and gives off carbon dioxide. Oxygenated blood then returns to the left atrium, passes through the left ventricle and is pumped out through the aorta to the systemic circulationwhere the oxygen is used and metabolized to carbon dioxide. In addition the blood carries nutrients from the digestive tract to various organs of the body, while transporting waste to the liver and kidneys. Normally with each heartbeat the right ventricle pumps the same amount of blood into the lungs as the left ventricle pumps to the body. Veins transport blood to the heart and carry deoxygenated blood – except for the pulmonary and portal veins. Arteries transport blood away from the heart, and apart from the pulmonary artery hold oxygenated blood. Their increased distance from the heart cause veins to have lower pressures than arteries. The heart contracts at a resting rate close to 72 beats per minute.Exercise temporarily increases the rate, but lowers resting heart rate in the long term, and is good for heart health.
Cardiovascular diseases (CVD) are the most common cause of death globally as of 2008, accounting for 30% of deaths. Of these more than three quarters follow coronary artery disease and stroke. Risk factors include: smoking, being overweight, little exercise, high cholesterol, high blood pressure, and poorly controlled diabetes, among others. Diagnosis of CVD is often done by listening to the heart-sounds with a stethoscope, ECG or by ultrasound. Specialists who focus on diseases of the heart are called cardiologists, although many specialties of medicine may be involved in treatment.
The human heart is situated in the middle mediastinum, at the level of thoracic vertebrae T5-T8. A double-membraned sac called the pericardium surrounds the heart and attaches to the mediastinum. The back surface of the heart lies near the vertebral column, and the front surface sits behind to the sternum and rib cartilages. The upper part of the heart is the attachment point for several large blood vessels – the venae cavae, aorta and pulmonary trunk. The upper part of the heart is located at the level of the third costal cartilage. The lower tip of the heart, the apex, lies to the left of the sternum (8 to 9cm from the midsternal line) between the junction of the fourth and fifth ribs near their articulation with the costal cartilages.
The largest part of the heart is usually slightly offset to the left side of the chest (though occasionally it may be offset to the right) and is felt to be on the left because the left heart is stronger and larger, since it pumps to all body parts. Because the heart is between the lungs, the left lung is smaller than the right lung and has a cardiac notch in its border to accommodate the heart. The heart is cone-shaped, with its base positioned upwards and tapering down to the apex. An adult heart has a mass of 250350 grams (912oz). The heart is typically the size of a fist: 12cm (5in) in length, 8cm (3.5in) wide, and 6cm (2.5in) in thickness. Well-trained athletes can have much larger hearts due to the effects of exercise on the heart muscle, similar to the response of skeletal muscle.
The heart has four chambers, two upper atria, the receiving chambers, and two lower ventricles, the discharging chambers. The atria open into the ventricles via the atrioventricular valves, present in the atrioventricular septum. This distinction is visible also on the surface of the heart as the coronary sulcus. There is an ear-shaped structure in the upper right atrium called the right atrial appendage, or auricle, and another in the upper left atrium, the left atrial appendage. The right atrium and the right ventricle together are sometimes referred to as the right heart. Similarly, the left atrium and the left ventricle together are sometimes referred to as the left heart. The ventricles are separated from each other by the interventricular septum, visible on the surface of the heart as the anterior longitudinal sulcus and the posterior interventricular sulcus.
The cardiac skeleton is made of dense connective tissue and this gives structure to the heart. It forms the atrioventricular septum which separates the atria from the ventricles, and the fibrous rings which serve as bases for the four heart valves. The cardiac skeleton also provides an important boundary in the heart’s electrical conduction system since collagen cannot conduct electricity. The interatrial septum separates the atria and the interventricular septum separates the ventricles. The interventricular septum is much thicker than the interatrial septum, since the ventricles need to generate greater pressure when they contract.
The heart, showing valves, arteries and veins. The white arrows shows the normal direction of blood flow.
The heart has four valves, which separate its chambers. The valves ensure blood flows in the correct direction through the heart and prevents backflow. Each valve consists of two to three cusps. The valves between the atria and ventricles connected to cartilaginous strings called chordae tendinae which in turn connect to muscles on the heart wall called papillary muscles.
The valves between the atria and ventricles are called the atrioventricular valves. Between the right atrium and the right ventricle is the tricuspid valve. The tricuspid valve has three cusps, which connect to chordae tendinae and three papillary muscles named the anterior, posterior, and septal muscles, after their relative positions. The mitral valve lies between the left atrium and left ventricle. It is also known as the bicuspid valve due to its having two cusps, an anterior and a posterior cusp. These cusps are also attached via chordae tendinae to two papillary muscles projecting from the ventricular wall.
The papillary muscles extends from the walls of the heart to the chordae tendinae of valves. These muscles prevent the valves from falling too far back when they close.During the relaxation phase of the cardiac cycle, the papillary muscles are also relaxed and the tension on the chordae tendineae is slight. As the heart chambers contract, so do the papillary muscles. This creates tension on the chordae tendineae, helping to hold the cusps of the atrioventricular valves in place and preventing them from being blown back into the atria.[g]
Two additional semilunar valves sit at the exit of each of the ventricles. The pulmonary valve is located at the base of the pulmonary artery. This has three cusps which are not attached to any papillary muscles. When the ventricle relaxes blood flows back into the ventricle from the artery and this flow of blood fills the pocket-like valve, pressing against the cusps which close to seal the valve. The semilunar aortic valve is at the base of the aorta and also is not attached to papillary muscles. This too has three cusps which close with the pressure of the blood flowing back from the aorta.
The right heart consists of two chambers, the right atrium and the right ventricle, separated by a valve, the tricuspid valve.
The right atrium receives blood almost continuously from the body’s two major veins, the superior and inferior venae cavae. A small amount of blood from the coronary circulation also drains into the right atrium via the coronary sinus, which is immediately above and to the middle of the opening of the inferior vena cava. In the wall of the right atrium is an oval-shaped depression known as the fossa ovalis, which is a remnant of an opening in the fetal heart known as the foramen ovale. Most of the internal surface of the right atrium is smooth, the depression of the fossa ovalis is medial, and the anterior surface has prominent ridges of pectinate muscles, which are also present in the right atrial appendage.
The right atrium is connected to the right ventricle by the tricuspid valve. The walls of the right ventricle are lined with trabeculae carneae, ridges of cardiac muscle covered by endocardium. In addition to these muscular ridges, a band of cardiac muscle, also covered by endocardium, known as the moderator band reinforces the thin walls of the right ventricle and plays a crucial role in cardiac conduction. It arises from the lower part of the interventricular septum and crosses the interior space of the right ventricle to connect with the inferior papillary muscle. The right ventricle tapers into the pulmonary trunk, into which it ejects blood when contracting. The pulmonary trunk branches into the left and right pulmonary arteries that carry the blood to each lung. The pulmonary valve lies between the right lung and the pulmonary trunk.
The left heart has two chambers: the left atrium, and the left ventricle, separated by the mitral valve.
The left atrium receives oxygenated blood back from the lungs via one of the four pulmonary veins. The left atrium has an outpouching called the left atrial appendage. Like the right atrium, the left atrium is lined by pectinate muscles. The left atrium is connected to the left ventricle by the mitral valve.
The left ventricle is much thicker as compared with the right, due to the greater force needed to pump blood to the entire body. Like the right ventricle, the left also has trabeculae carneae, but there is no moderator band. The left ventricle pumps blood to the body through the aortic valve and into the aorta. Two small openings above the aortic valve carry blood to the heart itself, the left main coronary artery and the right coronary artery.
The heart wall is made up of three layers: the inner endocardium, middle myocardium and outer epicardium. These are surrounded by a double-membraned sac called the pericardium.
The innermost layer of the heart is called the endocardium. It is made up of a lining of simple squamous epithelium, and covers heart chambers and valves. It is continuous with the endothelium of the veins and arteries of the heart, and is joined to the myocardium with a thin layer of connective tissue. The endocardium, by secreting endothelins, may also play a role in regulating the contraction of the myocardium.
The middle layer of the heart wall is the myocardium, which is the cardiac muscle a layer of involuntary striated muscle tissue surrounded by a framework of collagen. The cardiac muscle pattern is elegant and complex, as the muscle cells swirl and spiral around the chambers of the heart, with the outer muscles forming a figure 8 pattern around the atria and around the bases of the great vessels, and inner muscles formining a figure 8 around the two ventricles and proceed toward the apex. This complex swirling pattern allows the heart to pump blood more effectively.
There are two types of cells in cardiac muscle: muscle cells which have the ability to contract easily, and pacemaker cells of the conducting system. The muscle cells make up the bulk (99%) of cells in the atria and ventricles. These contractile cells are connected by intercalated discs which allow a rapid response to impulses of action potential from the pacemaker cells. The intercalated discs allow the cells to act as a syncytium and enable the contractions that pump blood through the heart and into the major arteries. The pacemaker cells make up 1% of cells and form the conduction system of the heart. They are generally much smaller than the contractile cells and have few myofibrils which gives them limited contractibility. Their function is similar in many respects to neurons. Cardiac muscle tissue has autorhythmicity, the unique ability to initiate a cardiac action potential at a fixed rate spreading the impulse rapidly from cell to cell to trigger the contraction of the entire heart.
The pericardium surrounds the heart. It consists of two membranes: an inner serous membrane called the epicardium, and an outer fibrous membrane. Blood vessels and nerves reach the cardiac muscle from the epicardium. These help influence the heart rate. These enclose the pericardial cavity which contains the pericardial fluid that lubricates the surface of the heart.
Heart tissue, like all cells in the body, need to be supplied with oxygen, nutrients and a way of removing metabolic wastes. This is achieved by the coronary circulation, which includes arteries, veins, and lymphatic vessels, Blood circulates through the coronary circulation cyclically, in peaks and troughs relating to the heart muscle’s relaxation or contraction.
Heart tissue receives blood from two arteries which arise just above the aortic valve. These are the left main coronary artery and the right coronary artery. The left main coronary artery splits shortly after leaving the aorta into two vessels, the left anterior descending and the left circumflex artery. The left anterior descending artery supplies heart tissue and the front, outer side, and the septum of the left ventricle. It does this by smaller branching arteries – diagonal and septal branches. The left circumflex supplies the back and underneath of the left ventricle. The right coronary artery supplies the right atrium, right ventricle, and lower posterior sections of the left ventricle. The right coronary artery also supplies blood to the atrioventricular node (in about 90% of people) and the sinoatrial node (in about 60% of people). The right coronary artery runs in a groove at the back of the heart and the left anterior descending artery runs in a groove at the front. There is significant variation between people in the anatomy of the arteries that supply the heart The arteries divide at their furtherst reaches into smaller branches that join together at the edges of each arterial distribution.
The coronary sinus is a large vein that drains into the right atrium, and receives most of the venous drainage of the heart. It receives blood from the great cardiac vein (receiving the left atrium and both ventricles), the posterior cardiac vein (draining the back of the left ventricle), the middle cardiac vein (draining the bottom of the left and right ventricles), and small cardiac veins. The anterior cardiac veins drain the front of the right ventricle and drain directly into the right atrium.
Small lymphatic networks called plexuses exist beneath each of the three layers of the heart. These networks collect into a main left and a main right trunk, which travel up the groove between the ventricles that exists on the heart’s surface, receiving smaller vessels as they travel up. These vessels then travel into the atrioventricular groove, and receive a third vessel which drains the section of the left ventricle sitting on the diaphragm. The left vessel joins with this third vessel, and travels along the pulmonary artery and left atrium, ending in the inferior tracheobronchial node. The right vessel travels along the right atrium and the part of the right ventricle sitting on the diaphragm. It usually then travels in front of the ascending aorta and then ends in a brachiocephalic node.
The heart is the first functional organ to develop and starts to beat and pump blood at about three weeks into embryogenesis. This early start is crucial for subsequent embryonic and prenatal development.
The heart derives from splanchnopleuric mesenchyme in the neural plate which forms the cardiogenic region. Two endocardial tubes form here that fuse to form a primitive heart tube known as the tubular heart. Between the third and fourth week, the heart tube lengthens, and begins to fold to form an S-shape within the pericardium. This places the chambers and major vessels into the correct alignment for the developed heart. Further development will include the septa and valves formation and remodelling of the heart chambers. By the end of the fifth week the septa are complete and the heart valves are completed by the ninth week.
Before the fifth week, there is an opening in the fetal heart known as the foramen ovale. The foramen ovale allowed blood in the fetal heart to pass directly from the right atrium to the left atrium, allowing some blood to bypass the lungs. Within seconds after birth, a flap of tissue known as the septum primum that previously acted as a valve closes the foramen ovale and establishes the typical cardiac circulation pattern. A depression in the surface of the right atrium remains where the foramen ovale once walls, called the fossa ovalis.
The embryonic heart begins beating at around 22 days after conception (5 weeks after the last normal menstrual period, LMP). It starts to beat at a rate near to the mother’s which is about 7580 beats per minute (bpm). The embryonic heart rate then accelerates and reaches a peak rate of 165185 bpm early in the early 7th week (early 9th week after the LMP). After 9 weeks (start of the fetal stage) it starts to decelerate, slowing to around 145 (25) bpm at birth. There is no difference in female and male heart rates before birth.
The heart functions as a pump in the circulatory system to provide a continuous flow of blood throughout the body. This circulation consists of the systemic circulation to and from the body and the pulmonary circulation to and from the lungs. Blood in the pulmonary circulation exchanges carbon dioxide for oxygen in the lungs through the process of respiration. The systemic circulation then transports oxygen to the body and returns carbon dioxide and relatively deoxygenated blood to the heart for transfer to the lungs.
The right heart collects deoxygenated blood from two large veins, the superior and inferior venae cavae. Blood collects in the right and left atrium continuously. The superior vena cava drains blood from above the diaphragm and empties into the upper back part of the right atrium. The inferior vena cava drains the blood from below the diaphragm and empties into the back part of the atrium below the opening for the superior vena cava. Immediately above and to the middle of the opening of the inferior vena cava is the opening of the thin-walled coronary sinus. Additionally, the coronary sinus returns deoxygenated blood from the myocardium to the right atrium. The blood collects in the right atrium. When the right atrium contracts, the blood is pumped through the tricuspid valve into the right ventricle. As the right ventricle contracts, the tricuspid valve closes and the blood is pumped into the pulmonary trunk through the pulmonary valve. The pulmonary trunk divides into pulmonary arteries and progressively smaller arteries throughout the lungs, until it reaches capillaries. As these pass by alveoli carbon dioxide is exchanged for oxygen. This happens through the passive process of diffusion.
In the left heart, oxygenated blood is returned to the left atrium via the pulmonary veins. It is then pumped into the left ventricle through the mitral valve and into the aorta through the aortic valve for systemic circulation. The aorta is a large artery that branches into many smaller arteries, arterioles, and ultimately capillaries. In the capillaries, oxygen and nutrients from blood are supplied to body cells for metabolism, and exchanged for carbon dioxide and waste products. Capillary blood, now deoxygenated, travels into venules and veins that ultimately collect in the superior and inferior vena cavae, and into the right heart.
The cardiac cycle refers to a complete heartbeat which includes systole and diastole and the intervening pause. The cycle begins with contraction of the atria and ends with relaxation of the ventricles. Systole refers to contraction of the atria or ventricles of the heart contract. Diastole is when the atria or ventricles relax and fill with blood. The atria and ventricles work in concert, so in systole when the ventricles are contracting, the atria are relaxed and collecting blood. When the ventricles are relaxed in diastole, the atria contract to pump blood to the ventricles. This coordination ensures blood is pumped efficiently to the body.
At the beginning of the cardiac cycle, in early diastole, both the atria and ventricles are relaxed. Since blood moves from areas of high pressure to areas of low pressure, when the chambers are relaxed, blood will flow into the atria (through the coronary sinus and the pulmonary veins). As the atria begin to fill, the pressure will rise so that the blood will move from the atria into the ventricles. In late diastole the atria contract, pumping more blood into the ventricles. This causes a rise in pressure in the ventricles. As the ventricles reach systole, blood will be pumped into the pulmonary artery (right ventricle) or aorta (left ventricle).
When the atrioventricular valves (tricuspid and mitral) are open, during blood flow to the ventricles, the aortic and pulmonary valves are closed to prevent backflow into the ventricles. When the ventricular pressure is greater than the atrial pressure the tricuspid and mitral valves will shut. When the ventricles contract the pressure forces the aortic and pulmonary valves open. As the ventricles relax, the aortic and pulmonary valves will close in response to decreased pressure.
Cardiac output (CO) is a measurement of the amount of blood pumped by each ventricle (stroke volume) in one minute. This is calculated by multiplying the stroke volume (SV) by the beats per minute of the heart rate (HR). So that: CO = SV x HR. The cardiac output is normalized to body size through body surface area and is called the cardiac index.
The average cardiac output, using an average stroke volume of about 70mL, is 5.25 L/min, with a normal range of 4.08.0 L/min. The stroke volume is normally measured using an echocardiogram and can be influenced by the size of the heart, physical and mental condition of the individual, sex, contractility, duration of contraction, preload and afterload.
Preload refers to the filling pressure of the atria at the end of diastole, when they are at their fullest. A main factor is how long it takes the ventricles to fillif the ventricles contract faster, then there is less time to fill and the preload will be less. Preload can also be affected by a person’s blood volume. The force of each contraction of the heart muscle is proportional to the preload, described as the Frank-Starling mechanism. This states that the force of contraction is directly proportional to the initial length of muscle fiber, meaning a ventricle will contract more forcefully, the more it is stretched.
Afterload, or how much pressure the heart must generate to eject blood at systole, is influenced by vascular resistance. It can be influenced by narrowing of the heart valves (stenosis) or contraction or relaxation of the peripheral blood vessels.
The strength of heart muscle contractions controls the stroke volume. This can be influenced positively or negatively by agents termed inotropes. These can be either conditions or drugs. Positive inotropes that cause stronger contractions include high blood calcium and drugs such as Digoxin, which will act to stimulate the sympathetic nerves in the fight-or-flight response. Negative inotropes causing weaker contractions include high blood potassium, hypoxia, acidosis, and drugs such as beta blockers and calcium channel blockers.
The normal rhythmical heart beat, called sinus rhythm, is established by the sinoatrial node, the heart’s pacemaker. Here an electrical signal is created that travels through the heart, causing the heart muscle to contract.
The sinoatrial node is found in the upper part of the right atrium near to the junction with the superior vena cava. The electrical signal generated by the sinoatrial node travels through the right atrium in a radial way that is not completely understood. It travels to the left atrium via Bachmann’s bundle, such that both left and right atria contract together. The signal then travels to the atrioventricular node. This is found at the bottom of the right atrium in the atrioventricular septumthe boundary between the right atrium and the left ventricle. The septum is part of the cardiac skeleton, tissue within the heart that the electrical signal cannot pass through, which forces the signal to pass through the atrioventricular node only. The signal then travels along the bundle of His to left and right bundle branches through to the ventricles of the heart. In the ventricles the signal is carried by specialized tissue called the Purkinje fibers which then transmit the electric charge to the cardiac muscle.
The normal resting heart rate is called the sinus rhythm, created and sustained by the sinoatrial node, a group of pacemaking cells found in the wall of the right atrium. Cells in the sinoatrial node do this by creating an action potential. The cardiac action potential is created by the movement of specific electrolytes into and out of the pacemaker cells. The action potential then spreads to nearby cells.
When the sinoatrial cells are resting, they have a negative charge on their membranes. However a rapid influx of sodium ions causes the membrane’s charge to become positive. This is called depolarisation and occurs spontaneously. Once the cell has a sufficiently high charge, the sodium channels close and calcium ions then begin to enter the cell, shortly after which potassium begins to leave it. All the ions travel through ion channels in the membrane of the sinoatrial cells. The potassium and calcium only start to move out of and into the cell once it has a sufficiently high charge, and so are called voltage-gated. Shortly after this, the calcium channels close and potassium channels open, allowing potassium to leave the cell. This causes the cell to have a negative resting charge and is called repolarization. When the membrane potential reaches approximately 60 mV, the potassium channels close and the process may begin again.
The ions move from areas where they are concentrated to where they are not. For this reason sodium moves into the cell from outside, and potassium moves from within the cell to outside the cell. Calcium also plays a critical role. Their influx through slow channels means that the sinoatrial cells have a prolonged “plateau” phase when they have a positive charge. A part of this is called the absolute refractory period. Calcium ions also combine with the regulatory protein troponin C in the troponin complex to enable contraction of the cardiac muscle, and separate from the protein to allow relaxation.
The normal sinus rhythm of the heart, giving the resting heart rate, is influenced by the autonomic nervous system through sympathetic and parasympathetic nerves. These arise from two paired cardiovascular centres in the medulla oblongata.The vagus nerve of the parasympathetic nervous system acts to decrease the heart rate, and nerves from the sympathetic trunk act to increase the heart rate. These come together in the cardiac plexus near the base of the heart. Without parasympathetic input which normally predominates, the sinoatrial node would generate a heart rate of about 100 bpm.
The nerves from the sympathetic trunk emerge through the T1-T4 thoracic ganglia and travel to both the sinoatrial and atrioventricular nodes, as well as to the atria and ventricles. The ventricles are more richly innervated by sympathetic fibers than parasympathetic fibers. Sympathetic stimulation causes the release of the neurotransmitter norepinephrine (also known as noradrenaline) at the neuromuscular junction of the cardiac nerves. This shortens the repolarization period, thus speeding the rate of depolarization and contraction, which results in an increased heart rate. It opens chemical or ligand-gated sodium and calcium ion channels, allowing an influx of positively charged ions. Norepinephrine binds to the beta1 receptor. High blood pressure medications are used to block these receptors and so reduce the heart rate.
The cardiovascular centres receive input from a series of receptors including proprioreceptors, baroreceptors, and chemoreceptors, plus stimuli from the limbic system. Through a series of reflexes these help regulate and sustain blood flow. For example, increased physical activity results in increased rates of firing by various proprioreceptors located in muscles, joint capsules, and tendons. With increased rates of firing, the parasympathetic stimulation may decrease or sympathetic stimulation may increase as needed in order to increase blood flow.
Similarly, baroreceptors are stretch receptors located in the aortic sinus, carotid bodies, the venae cavae, and other locations, including pulmonary vessels and the right side of the heart itself. Rates of firing from the baroreceptors represent blood pressure, level of physical activity, and the relative distribution of blood. The cardiac centers monitor baroreceptor firing to maintain cardiac homeostasis, a mechanism called the baroreceptor reflex. With increased pressure and stretch, the rate of baroreceptor firing increases, and the cardiac centers decrease sympathetic stimulation and increase parasympathetic stimulation. As pressure and stretch decrease, the rate of baroreceptor firing decreases, and the cardiac centers increase sympathetic stimulation and decrease parasympathetic stimulation.
There is a similar reflex, called the atrial reflex or Bainbridge reflex, associated with varying rates of blood flow to the atria. Increased venous return stretches the walls of the atria where specialized baroreceptors are located. However, as the atrial baroreceptors increase their rate of firing and as they stretch due to the increased blood pressure, the cardiac center responds by increasing sympathetic stimulation and inhibiting parasympathetic stimulation to increase heart rate. The opposite is also true.
In addition to the autonomic nervous system, other factors can impact on this. These include epinephrine, norepinephrine, and thyroid hormones; levels of various ions including calcium, potassium, and sodium; body temperature; hypoxia; and pH balance. Factors that increase the heart rate can include release of norepinephrine, hypoxemia, low blood pressure and dehydration, a strong emotional response, a higher body temperature, and metabolic and hormonal factors such as a low potassium or sodium level or stimulus from thyroid hormones. Decreased body temperature, relaxation, and metabolic factors can also contribute to a decrease in heart rate.
The resting heart rate of a newborn can be 129 beats per minute (bpm) and this gradually decreases until maturity. The adult resting heart rate ranges from 60 to 100 bpm. Exercise and fitness levels, age and basal metabolic rate can all affect the heart rate. An athlete’s heart rate can be lower than 60 bpm. During exercise the rate can be 150 bpm with maximum rates reaching from 200 to 220 bpm.
One of the simplest methods of assessing the heart’s condition is to listen to it using a stethoscope. Typically, healthy hearts have only two audible heart sounds, called S1 and S2. The first heart sound S1, is the sound created by the closing of the atrioventricular valves during ventricular contraction and is normally described as “lub”. The second heart sound, S2, is the sound of the semilunar valves closing during ventricular diastole and is described as “dub”. Each sound consists of two components, reflecting the slight difference in time as the two valves close. S2 may split into two distinct sounds, either as a result of inspiration or different valvular or cardiac problems. Additional heart sounds may also be present and these give rise to gallop rhythms. A third heart sound, S3 usually indicates an increase in ventricular blood volume. A fourth heart sound S4 is referred to as an atrial gallop and is produced by the sound of blood being forced into a stiff ventricle. The combined presence of S3 and S4 give a quadruple gallop.
Heart murmurs are abnormal heart sounds which can be either pathological or benign. One example of a murmur is Still’s murmur, which presents a musical sound in children, has no symptoms and disappears in adolescence.
A different type of sound, a pericardial friction rub can be heard in cases of pericarditis where the inflamed membranes can rub together.
Cardiovascular diseases, which include diseases of the heart, are the leading cause of death worldwide. The majority of cardiovascular disease is noncommunicable and related to lifestyle and other factors, becoming more prevalent with ageing. Heart disease is a major cause of death, accounting for an average of 30% of all deaths in 2008, globally. This rate varies from a lower 28% to a high 40% in high-income countries. Doctors that specialise in the heart are called cardiologists. Many other medical professionals are involved in treating diseases of the heart, including doctors such as general practitioners, cardiothoracic surgeons and intensivists, and allied health practitioners including physiotherapists and dieticians.
Coronary artery disease is also known as ischemic heart disease, is caused by atherosclerosis a build-up of plaque along the inner walls of the arteries which narrows them, reducing the blood flow to the heart. A stable plaque may cause chest pain (angina) or breathlessness during exercise or at rest, or no symptoms at all. A ruptured plaque can block a blood vessel and lead to ischaemia of the heart muscle, causing unstable angina or a heart attack. In the worst case this may cause cardiac arrest, a sudden and utter loss of output from the heart.Obesity, high blood pressure, uncontrolled diabetes, smoking and high cholesterol can all increase the risk of developing atherosclerosis and coronary artery disease.
Heart failure is where the heart can’t beat enough blood to meet the needs of the body. It is generally a chronic condition, associated with age, that progresses gradually.Each side of the heart can fail independently of the other, resulting in heart failure of the right heart or the left heart. Left heart failure can also lead to right heart failure (cor pulmonale) by increasing strain on the right heart. If the heart is unable to pump sufficient blood, it may accumulate throughout the body, causing breathlessness in the lungs (pulmonary congestion; pulmonary edema), swelling (edema) of the feet or other gravity-dependent areas, decrease exercise tolerance, or cause other clinical signs such as an enlarged liver, cardiac murmurs, or a raised jugular venous pressure. Common causes of heart failure include coronary artery disease, valve disorders and diseases of cardiac muscle.
Cardiomyopathy is a noticeable deterioration of the heart muscle’s ability to contract, which can lead to heart failure. The causes of many types of cardiomyopathy are poorly understood; some identified causes include alcohol, toxins, systemic disease such as sarcoidosis, and congenital conditions such as HOCM. The types of cardiomyopathy are described according to how they affect heart muscle. Cardiomyopathy can cause the heart to become enlarged (hypertrophic cardiomyopathy), constrict the outflow tracts of the heart (restrictive cardiomyopathy), or cause the heart to dilate and impact on the effiency of its beating (dilated cardiomyopathy). HOCM is often undiagnosed and can cause sudden death in young athletes.
Heart murmurs are abnormal heart sounds which can be either related to disease or benign, and there are several kinds. There are normally two heart sounds, and abnormal heart sounds can either be extra sounds, or “murmurs” related to the flow of blood between the sounds. Murmurs are graded by volume, from 1) the quietest, to 6) the loudest, and evaluated by their relationship to the heart sounds, position in the cardiac cycle, and additional features such as their radiation to other sites, changes with a person’s position, the frequency of the sound as determined by the side of the stethoscope by which they are heard, and site at which they are heard loudest.Phonocardiograms can record these sounds, and echocardiograms are generally required for their diagnosis. Murmurs can result from valvular heart diseases due to narrowing (stenosis), or regurgitation of any of the main heart valves, such as aortic stenosis, mitral regurgitation or mitral valve prolapse. They can also result from a number of other disorders, including atrial and ventricular septal defects. Two common and infective causes of heart murmurs, are infective endocarditis and rheumatic fever, particularly in developing countries. Infective endocarditis involves colonisation of a heart valve, and rheumatic fever involves an initial bacterial infection by Group A streptococcus followed by a reaction against heart tissue that resembles the streptococcal antigen.
Abnormalities in the normal sinus rhythm of the heart can prevent the heart from effectively pumping blood, and are generally identified by ECG. These cardiac arrhythmias can cause an abnormal but regular heart rhythm, such as a rapid heart rate (tachycardia, classified as arising from above the ventricles or from the ventricles) or a slow heart rate (bradycardia); or may result in irregular rhythms. Tachycardia is generally defined as a heart rate faster than 100 beats per minute, and bradycardia as a heart rate slower than 60.Asystole is the cessation of heart rhythm. A random and varying rhythm is classified as atrial or ventricular fibrillation depending if the electrical activity originates in the atria or the ventricles. Abnormal conduction can cause a delay or unusual order of contraction of the heart muscle. This can be a result of a disease process, such as heart block, or congenital, such as Wolff-Parkinson-White syndrome.
Diseases may also affect the pericardium which surrounds the heart, which when inflammed is called pericarditis. This may result from infective causes (such as glandular fever, cytomegalovirus, coxsackievirus, tuberculosis or Q fever), systemic disorders such as amyloidosis or sarcoidosis, tumours, high uric acid levels, and other causes. This inflammation affects the ability of the heart to pump effectively. When fluid builds up in the pericardium this is called pericardial effusion, which when it causes acute heart failure is called cardiac tamponade. This may be blood from a traumatic injury or fluid from an effusion. This can compress the heart and adversely affect the function of the heart. The fluid can be removed from the pericardial sac using a syringe in a procedure called pericardiocentesis.
The heart can be affected by congenital diseases. These include failure of the developmental foramen ovale to close, present in up to 25% of people;ventricular or atrial septal defects, congenital diseases of the heart valves (e.g. congenital aortic stenosis) or disease relating to blood vessels or blood flow from the heart (such as a patent ductus arteriosus or aortic coarctation).; Harrisons 14581465 These may cause symptoms at a variety of ages. If unoxygenated blood travels directly from the right to the left side of the heart, it may be noticed at birth, as it may cause a baby to become blue (cyanotic) such as Tetralogy of Fallot. A heart problem may impact a child’s ability to grow. Some causes rectify with time and are regarded as benign. Other causes may be incidentally detected on a cardiac examination. These disorders are often diagnosed on an echocardiogram.
Heart disease is diagnosed by the taking of a medical history, a cardiac examination, and further investigations, including blood tests, echocardiograms, ECGs and imaging. Other invasive procedures such as cardiac catheterisation can also play a role.
The cardiac examination includes inspection, feeling the chest with the hands (palpation) and listening with a stethoscope (auscultation). It involves assessment of signs that may be visible on a person’s hands (such as splinter haemorrhages), joints and other areas. A person’s pulse is taken, usually at the radial artery near the wrist, in order to assess for the rhythm and strength of the pulse. The blood pressure is taken, using either a manual or automatic sphygmomanometer or using a more invasive measurement from within the artery. Any elevation of the jugular venous pulse is noted. A person’s chest is felt for any transmitted vibrations from the heart, and then listened to with a stethoscope. A normal heart has two hearts sounds – additional heart sounds or heart murmurs may also be able to be heard and may point to disease. Additional tests may be conducted to assess a person’s heart murmurs if they are present, and peripheral signs of heart disease such as swollen feet or fluid in the lungs may be assessed.
Using surface electrodes on the body, it is possible to record the electrical activity of the heart. This tracing of the electrical signal is the electrocardiogram (ECG) or (EKG). An ECG is a bedside test and usually requires the placement of ten leads on the body. This produces a “12 lead” ECG (three extra leads are calculated mathematically, and one lead is a ground).
There are five prominent features on the ECG: the P wave (atrial depolarisation), the QRS complex (atrial repolarisation and ventricular depolarisation) and the T wave (ventricular repolarisation). These reflect the summed action potential of the heart’s muscle cells as they contract. A downward deflection on the ECG implies cells are becoming more negative in charge (“depolarising”), whereas an upward inflection implies cells are becoming more positive (“repolarising”). The ECG is a useful tool in detecting rhythm disturbances and in detecting insufficient blood supply to the heart. Sometimes abnormalities are not immediately visible on the ECG. Testing when exercising can be used to provoke an abnormality, or an ECG can be worn for a longer period such as a 24-hour Holter monitor if a suspected rhythm abnormality is not present at the time of assessment.
Several imaging methods can be used to assess the anatomy and function of the heart, including ultrasound (echocardiography), angiography, CT scans, MRI and PET. An echocardiogram is an ultrasound of the heart used to measure the heart’s function, assess for valve disease, and look for any abnormalities. Echocardiography can be conducted by a probe on the chest (“transthoracic”) or by a probe in the esophagus (“transoesophageal”). A typical echocardiography report will include information about the width of the valves noting any stenosis, whether there is any backflow of blood (regurgitation) and information about the blood volumes at the end of systole and diastole, including an ejection fraction, which describes how much blood is ejected from the left and right ventricles after systole. Ejection fraction can then be obtained by dividing the volume ejected by the heart (stroke volume) by the volume of the filled heart (end-diastolic volume). Echocardiograms can also be conducted under circumstances when the body is more stressed, in order to examine for signs of lack of blood supply. This cardiac stress test involves either direct exercise, or where this is not possible, injection of a drug such as dobutamine.
CT scans, chest X-rays and other forms of imaging can help evaluate the heart’s size, evaluate for signs of pulmonary oedema, and indicate whether there is fluid around the heart. They are also useful for evaluating the aorta, the major blood vessel which leaves the heart.
A number of medications are used to treat diseases of the heart, or ameliorate symptoms.
For diseases of the heart rate or rhythm, a number of different antiarrhythmic agents are used. These may interfere with electrolyte channels and thus the cardiac action potential (such as calcium channel blockers, sodium channel blockers), interfere with stimulation of the heart by the sympathetic nervous system (beta blockers), or interfere with the movement of sodium and potassium across the cell membrane, such as digoxin. Other examples include atropine for slow rhythms, and amiodarone for irregular rhythms. Such medications are not the only way of treating diseases of heart rate or rhythm. In the context of a new-onset irregular heart rhythm (atrial fibrillation), immediate electrical cardioversion may be attempted. For a slow heartbeat or heart block, a pacemaker or defibrillator may be inserted. The acuity of onset often affects how a rhythm disturbance is managed, as does whether a rhythm causes hemodynamic instability, such as low blood pressure or symptoms. An instigating cause is investigated for, such as a heart attack, medication, or metabolic problem.
For ischaemic heart disease, treatment also includes amelioration of symptoms. This includes GTN, beta blockers and, in the context of an acute event, stronger pain relief such as morphine and other opiates. Many of these drugs have additional protective benefits, by decreasing the sympathetic tone on the heart that occurs with the pain, or by dilating blood vessels (GTN).
Treatment of heart disease includes primary and secondary prevention to prevent the occurrence or worsening of symptoms and atherosclerosis. This includes recommendations to cease smoking, decrease alcohol consumption, increase exercise, and make modifications to their diet to decrease the consumption of fats and sugars. Medications may also be given to help better control concurrent diabetes. Statins or other drugs such as fibrates may also be given to decrease a person’s cholesterol levels. Blood pressure medication may also be commenced or modified.
For many diseases of the heart, including atrial fibrillation and valvular disease, and after a heart operation, anticoagulation in the form of aspirin, warfarin, clopidogrel or novel oral anticoagulants is often given simultaneously, because of an increased risk of stroke or, in the context of a clotted heart vessel, rethrombosis.
Surgery, when considered necessary for diseases of the heart, can take place via an open operation or via small guidewires inserted via peripheral arteries (“percutaneous coronary intervention”). Percutaneous coronary intervention is usually used in the context of an acute coronary syndrome, and may be used to insert a stent.
Coronary artery bypass surgery is one such operation. In this operation, one or more arteries surrounding the heart that have become narrowed are bypassed. This is done by taking blood vessels harvested from another part of the body. Commonly harvested veins include the saphenous veins or the internal mammary artery. Because this operation involves the heart tissue, a machine is used so that blood can bypass the heart during the operation.
Heart valve repair or valve replacement are options for diseases of the heart valves.
Humans have known about the heart since ancient times, although its precise function and anatomy were not clearly understood. From the primarily religious views of earlier societies towards the heart, ancient Greeks are considered to have been the primary seat of scientific understanding of the heart in the ancient world. Aristotle considered the heart to be organ responsible for creating blood; Plato considered the heart as the source of circulating blood and Hippocrates noted blood circulating cyclically from the body through the heart to the lungs.Erasistratos (304-250 BC) noted the heart as a pump, causing dilation of blood vessels, and noted that arteries and veins both radiate from the heart, becoming progressively smaller with distance, although he believed they were filled with air and not blood. He also discovered the heart valves.
The Greek physician Galen (2nd century AD) knew blood vessels carried blood and identified venous (dark red) and arterial (brighter and thinner) blood, each with distinct and separate functions. Galen, noting the heart as the hottest organ in the body, concluded that it provided heat to the body. The heart did not pump blood around, the heart’s motion sucked blood in during diastole and the blood moved by the pulsation of the arteries themselves.  Galen believed the arterial blood was created by venous blood passing from the left ventricle to the right through ‘pores’ between the ventricles. Air from the lungs passed from the lungs via the pulmonary artery to the left side of the heart and created arterial blood. 
These ideas went unchallenged for almost a thousand years.
The earliest descriptions of the coronary and pulmonary circulation systems can be found in the Commentary on Anatomy in Avicenna’s Canon, published in 1242 by Ibn al-Nafis. In his manuscript, al-Nafis wrote that blood passes through the pulmonary circulation instead of moving from the right to the left ventricle as previously believed by Galen. His work was later translated into Latin by Andrea Alpago.
In Europe, the teachings of Galen continued to dominate the academic community and his doctrines were adopted as the official canon of the Church. Andreas Vesalius questioned some of Galen’s beliefs of the heart in De humani corporis fabrica (1543), but his magnum opus was interpreted as a challenge to the authorities and he was subjected to a number of attacks.Michael Servetus wrote in Christianismi Restitutio (1553) that blood flows from one side of the heart to the other via the lungs.
The breakthrough came with the publication of De Motu Cordis (1628) by the English physician William Harvey. Harvey’s book completely describes the systemic circulation and the mechanical force of the heart, leading to an overhaul of the Galenic doctrines.Otto Frank (18651944) was a German physiologist; among his many published works are detailed studies of this important heart relationship. Ernest Starling (18661927) was an important English physiologist who also studied the heart. Although they worked largely independently, their combined efforts and similar conclusions have been recognized in the name “FrankStarling mechanism”.
Although Purkinje fibers and the bundle of His were discovered as early as the 19th century, their specific role in the electrical conduction system of the heart remained unknown until Sunao Tawara published his monograph, titled Das Reizleitungssystem des Sugetierherzens, in 1906. Tawara’s discovery of the atrioventricular node prompted Arthur Keith and Martin Flack to look for similar structures in the heart, leading to their discovery of the sinoatrial node several months later. These structures form the anatomical basis of the electrocardiogram, whose inventor, Willem Einthoven, was awarded the Nobel Prize in Medicine or Physiology in 1924.
The first successful heart transplantation was performed in 1967 by the South African surgeon Christiaan Barnard at Groote Schuur Hospital in Cape Town. This marked an important milestone in cardiac surgery, capturing the attention of both the medical profession and the world at large. However, long-term survival rates of patients were initially very low. Louis Washkansky, the first recipient of a donated heart, died 18 days after the operation while other patients did not survive for more than a few weeks. The American surgeon Norman Shumway has been credited for his efforts to improve transplantation techniques, along with pioneers Richard Lower, Vladimir Demikhov and Adrian Kantrowitz. As of March 2000, more than 55,000 heart transplantations have been performed worldwide.
By the middle of the 20th century, heart disease had surpassed infectious disease as the leading cause of death in the United States, and it is currently the leading cause of deaths worldwide. Since 1948, the ongoing Framingham Heart Study has shed light on the effects of various influences on the heart, including diet, exercise, and common medications such as aspirin. Although the introduction of ACE inhibitors and beta blockers has improved the management of chronic heart failure, the disease continues to be an enormous medical and societal burden, with 30 to 40% of patients dying within a year of receiving the diagnosis.
As one of the vital organs, the heart was long identified as the center of the entire body, the seat of life, or emotion, or reason, will, intellect, purpose or the mind. The heart is an emblematic symbol in many religions, signifying “truth, consience or moral courage in many religions – the temple or throne of God in Islamic and Judeo-Christian thought; the divine centre, or atman, and the third eye of transcendent wisdom in Hinduism; the diamond of purity and essence of the Buddha; the Taoist centre of understanding.”
In the Hebrew Bible, the word for heart, lev, is used in these meanings, as the seat of emotion, the mind, and referring to the anatomical organ. It is also connected in function and symbolism to the stomach.
An important part of the concept of the soul in Ancient Egyptian religion was thought to be the heart, or ib. The ib or metaphysical heart was believed to be formed from one drop of blood from the child’s mother’s heart, taken at conception. To ancient Egyptians, the heart was the seat of emotion, thought, will, and intention. This is evidenced by Egyptian expressions which incorporate the word ib, such as Awi-ib for “happy” (literally, “long of heart”), Xak-ib for “estranged” (literally, “truncated of heart”). In Egyptian religion, the heart was the key to the afterlife. It was conceived as surviving death in the nether world, where it gave evidence for, or against, its possessor. It was thought that the heart was examined by Anubis and a variety of deities during the Weighing of the Heart ceremony. If the heart weighed more than the feather of Maat, which symbolized the ideal standard of behavior. If the scales balanced, it meant the heart’s possessor had lived a just life and could enter the afterlife; if the heart was heavier, it would be devoured by the monster Ammit.
The Chinese character for “heart”, , derives from a comparatively realistic depiction of a heart (indicating the heart chambers) in seal script. The Chinese word xn also takes the metaphorical meanings of “mind”, “intention”, or “core”.In Chinese medicine, the heart is seen as the center of shn “spirit, consciousness”. The heart is associated with the small intestine, tongue, governs the six organs and five viscera, and belongs to fire in the five elements.
The Sanskrit word for heart is hd or hdaya, found in the oldest surviving Sanskrit text, the Rigveda. In Sanskrit, it may mean both the anatomical object and “mind” or “soul”, representing the seat of emotion. Hrd may be a cognate of the word for heart in Greek, Latin, and English.
Many classical philosophers and scientists, including Aristotle, considered the heart the seat of thought, reason, or emotion, often disregarding the brain as contributing to those functions. The identification of the heart as the seat of emotions in particular is due to the Roman physician Galen, who also located the seat of the passions in the liver, and the seat of reason in the brain.
The heart also played a role in the Aztec system of belief. The most common form of human sacrifice practiced by the Aztecs was heart-extraction. The Aztec believed that the heart (tona) was both the seat of the individual and a fragment of the Sun’s heat (istli). To this day, the Nahua consider the Sun to be a heart-soul (tona-tiuh): “round, hot, pulsating”.
In Catholicism, there has been a long tradition of worship of the heart, stemming from worship of the wounds of Jesus Christ which gained prominence from the mid sixteenth century. This tradition influenced the development of the medieval Christian devotion to the Sacred Heart of Jesus and the parallel worship of Immaculate Heart of Mary, made popular by John Eudes.
The expression of a broken heart is a cross-cultural reference to grief for a lost one or to unfulfilled romantic love.
The notion of “Cupid’s arrows” is ancient, due to Ovid, but while Ovid describes Cupid as wounding his victims with his arrows, it is not made explicit that it is the heart that is wounded. The familiar iconography of Cupid shooting little heart symbols is a Renaissance theme that became tied to Valentine’s day.
Animal hearts are widely consumed as food. As they are almost entirely muscle, they are high in protein. They are often included in dishes with other offal, for example in the pan-Ottoman kokoretsi.
Heart – Wikipedia, the free encyclopedia
Embryonic stem cells seen through a microscope. The study saw a 40% reduction in the size of scarred tissue on the patients hearts. Photograph: Mauricio Lima/AFP/Getty Images
People suffering from heart disease have been offered hope by a new study that suggests damaged tissue could be regenerated through a stem cell treatment injected into the heart during surgery.
The small-scale study, published in the Journal of Cardiovascular Translational Research, followed 11 patients who during bypass surgery had stem cells injected into their hearts near the site of tissue scars caused by heart attacks.
One of the trials most dramatic results was a 40% reduction in the size of scarred tissue. Such scarring occurs during a cardiac event such as a heart attack, and can increase the chances of further heart failure. The scarring was previously thought to be permanent and irreversible.
At the time of treatment, the patients were suffering heart failure and had a very high (70%) annual mortality rate. But 36 months after receiving the stem cell treatment all are still alive, and none have suffered a further cardiac event such as a heart attack or stroke, or had any readmissions for cardiac-related reasons.
According to the British Heart Foundation, while there are several treatments to help people with heart failure, there is no known cure, and in some cases a heart transplant may be the only option.
Twenty-four months after participants were injected with the stem cell treatment there was a 30% improvement in heart function, 40% reduction in scar size, and 70% improvement in quality of life, as judged by the Minnesota living with heart failure (MLHF) score.
Related: Brain damage could be repaired by creating new nerve cells
Quite frankly it was a big surprise to find the area of scar in the damaged heart got smaller, said Prof Stephen Westaby from John Radcliffe hospital in Oxford, who undertook the research at AHEPA university hospital in Thessaloniki, Greece, with Kryiakos Anastasiadis and Polychronis Antonitsis.
Westaby began theorising about the impact of stem cells on regenerating heart tissue and reducing scarring after observing how scar tissue on the hearts of babies who have had heart attacks and undergone heart failure disappeared by the time they reached adolescence, suggesting that residual stem cells might be able to repair the damaged tissue.
Its an early study and its difficult to make large-scale predictions based on small studies, said Ajan Reginald, the founder of Celixir, the company that produces the treatment. But even in a small study you dont expect to see results this dramatic.
These are 11 patients who were in advanced heart failure, they had had a heart attack in the past, multiple heart attacks in many cases. The life expectancy for these patients is less than two years, were excited and honoured that these patients are still alive.
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Jeremy Pearson, the associate medical director at the British Heart Foundation (BHF), said: This very small study suggests that targeted injection into the heart of carefully prepared cells from a healthy donor during bypass surgery, is safe. It is difficult to be sure that the cells had a beneficial effect because all patients were undergoing bypass surgery at the same time, which would usually improve heart function.
A controlled trial with substantially more patients is needed to determine whether injection of these types of cells proves any more effective than previous attempts to improve heart function in this way, which have so far largely failed.
Westaby conceded that the improvement in patients health was partly due to the heart bypass surgery those in the study were undergoing, and said the next study would include a control group who undergo bypass but do not receive stem cell treatment, to measure exactly what impact the treatment has.
These patients came out of heart failure partly due to the bypass grafts of course, but we think it was partly due to the fact that they had a smaller area of scar [as a result of the stem cell treatment]. Certainly this finding of scar being reduced is quite fascinating, he said.
Westaby will commence a large-scale controlled study later this year at the Royal Brompton hospital in London, and Celixir hopes to make the Heartcel treatment available to patients in 2018 or 2019.
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Stem cell trial suggests damaged heart tissue could be …
Cells Weekly is a digest of the most interesting news and events in stem cell research, cell therapy and regenerative medicine. Cells Weekly is posted every Sunday night!
1. Proliferation of unproved stem cell clinics in US The biggest buzz this week was a study by @LeighGTurner and @pknoepfler on stem cell clinics in US. This is very interesting study, which Id highly recommend you to read! Forget about stem cell tourism, all you need to do now is to look across the street or open glossy magazine at airplane. They are everywhere:
Using rigorous Internet-based key word searches (see Supplemental Information for details), we found 351 U.S. businesses engaged in direct-to-consumer marketing of stem cell interventions offered at 570 clinics. For each business, we collected the company name, location(s), website address, advertised stem cell types, and diseases, injuries, and other conditions that clinics claim to treat with stem cell interventions. (Table S1 lists and describes all of the businesses we identified).
The authors did a lot of work and they are also open to suggestions for clinics database and improvements in methodology. Also read about this study on the Niche blog.
2. Failure of Phase 3 cardiac cell therapy trial This week Belgian company Celyad released headline results of their Phase 3 cardiac cell therapy pan-European trial CHART-1. It was efficacy assessment of auto- bone marrow MSC, induced in cardiac lineage, on 271 patients with chronic ischemic heart failure. Primary endpoints of the study were missed. So, study is failed. However, post-hoc analysis showed 60% of patient subgroup with significant efficacy, measured by primary endpoint. Companys press release has a positive spin and most media outlets called the results of the trial mixed or even promising. The fate of so-called C-CURE cell therapy now will depend on discussion with EMA in Europe. Company still may attempt to commercialize it and/ or continue with re-designed trial in US. In any case Celyad would not proceed with C-CURE without partner. Unfortunately for Celyad, dropping of the companys value by investors for more than a third, will make a search for potential partner very hard.
3. Stem cell professionals divided for 2 camps in best regulation debate Article in STAT nicely summarized recent debate on the best rergulatory framework for stem cell-based therapies in US. In the first camp, proponents of so-called Regrow Act and anyone else (for example, president of CIRM, Randy Mills), who supports reformation of FDA to accelerate approvals and ease regulation in general. In the second camp, professionals, who are opposing simple ease of current FDA regulation. In this camp, we can see such professional organizations as ISSCR, ARM and some patient advocacy groups. As the example of second camp, you can read Knoepflers op-ed in the San Francisco Chronicle. Very interesting debate! Let me know what do you think in comments.
4. More clinical trials updates The results of the Phase 2 ALS trial, sponsored by NeuralStem were published online in Neurology on June 29, 2016. 15 patients, divided for 5 treatments groups, underwent experimental procedures in 3 different US centers. Even though there were 2 cases of serious adverse events observed and 2 deaths (attributed to disease progression) before 270 days, procedure deemed to be safe and well tolerated in general. Important conclusion experimental treatment was not clinically beneficial. However, as it was highlighted by investigators, the trial was not designed and powered to assess efficacy.
Last year, US-based company Cytori had 2 cardiac cell therapy trials, called ATHENA 1 and 2. Both trials were terminated last year, due to safety concerns and business considerations. Recently, company published available data, generated from both trials. 31 patients were included in analysis (28 from ATHENA 1 and 3 from ATHENA 2) 17 in cell therapy group and 14 in placebo group. Patients with chronic myocardial ischemia received 40 or 80 millions of autologous adipose tissue-derived stromal vascular fraction cells, processed from lipoaspirates at point of care with Celution system. Serious adverse events in 2 patients (related to procedure, but not related to cells) triggered stopping rule and studies were suspended. FDA allowed to continue after amendment, however company decided to terminate trials. In relation to safety, ATHENA 1 did not meet primary endpoint, measured by major adverse cardiac events (MACE) 35.3% in cell therapy group versus 21.4% in placebo. Some efficacy endpoints, specified in ATHENA 2 were met at some time points. The authors concluded that studies were feasible with suggestion of benefit.
5. Caution about new gene therapy trials Two recent proposals for new gene therapy trials in US, evaluated by NIH Recombinant DNA Advisory Committee (RAC) caused concerns about safety. Nature editorial this week covered proposals of Dimension Therapeutics and University of Pennsylvania, saying it must proceed with caution. Links about proposal for the first CRISPR-based application in human, you can look in previous Cells Weekly. Here is decision on Dimensions proposal:
After some discussion, the RAC voted unanimously to approve the trial. However, that came with a long list of conditions, including that the treatment first be tested in a second animal species. The researchers disagree with most of the conditions, believing that more expensive animal trials will add nothing. They feel that they are being held to a different standard from most trials. Dimension still plans to submit an application to the US Food and Drug Administration (FDA) later this year to start a clinical trial. It is unclear how heavily the RACs recommendations weigh into FDA decisions, but Wadsworth says that the company will conduct its trials overseas if necessary. These patients have been waiting a long time, he says.
6. Is FDA really holding stem cell therapy developers? Based on the recent post on California Stem Cell Report, the answer is YES. This opinion was voiced by CIRMs president Randy Mills several times. Ive asked Jan Nolta (IND submitter) on twitter FDA request of $330k pigs experiments and she said that it makes sense to do and $330k was misquoted.
Do you guys have any good examples when FDA was unreasonably holding developers with their INDs and trials progression? Please comment!
7. MSC and cancer friends or foes? New interesting data came up this week about impact of MSC-based therapy on carcinogenesis. Researchers found that in breast tumor model coinjection and distant injection of MSC has different impact on tumor growth:
Unlike previous reports, this is the first study reporting that MSCs may exert opposite roles on tumor growth in the same animal model by modulating the host immune system, which may shed light on the potential application of MSCs as vehicles for tumor therapy and other clinical applications.
8. New methods and protocols: Osteogenic differentiation of bioprinted constructs consisting of human adipose-derived stem cells (PLoS ONE) Bone marrow is a reservoir for cardiac resident stem cells (Sci Rep) Multiple genetically engineered humanized microenvironments in a single mouse (Biomater Res) Xenotransplantation of human fetal cardiac progenitor cells is useless in porcine model of ischemic heart failure (PLoS ONE) Feeding strategies in expansion of human pluripotent stem cells in stirred tank bioreactors (Stem Cells TM)
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Stem Cell Assays Reproducible Research on Stem Cells
There are several ways adult stem cells can be isolated, most of which are being actively explored by our researchers.
1) From the body itself: Scientists are discovering that many tissues and organs contain a small number of adult stem cells that help maintain them. Adult stem cells have been found in the brain, bone marrow, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, and other (although not all) organs and tissues. They are thought to live in a specific area of each tissue, where they may remain dormant for years, dividing and creating new cells only when they are activated by tissue injury, disease or anything else that makes the body need more cells.
Adult stem cells can be isolated from the body in different ways, depending on the tissue. Blood stem cells, for example, can be taken from a donors bone marrow, from blood in the umbilical cord when a baby is born, or from a persons circulating blood. Mesenchymal stem cells, which can make bone, cartilage, fat, fibrous connective tissue, and cells that support the formation of blood can also be isolated from bone marrow. Neural stem cells (which form the brains three major cell types) have been isolated from the brain and spinal cord. Research teams at Childrens, headed by leading scientists Stuart Orkin, MD and William Pu, MD, both affiliate members of the Stem Cell Program, recently isolated cardiac stem cells from the heart.
Isolating adult stem cells, however, is just the first step. The cells then need to be grown to large enough numbers to be useful for treatment purposes. The laboratory of Leonard Zon, MD, director of the Stem Cell Program, has developed a technique for boosting numbers of blood stem cells thats now in Phase I clinical testing.
2) From amniotic fluid: Amniotic fluid, which bathes the fetus in the womb, contains fetal cells including mesenchymal stem cells, which are able to make a variety of tissues. Many pregnant women elect to have amniotic fluid drawn to test for chromosome defects, the procedure known as amniocentesis. This fluid is normally discarded after testing, but Childrens Hospital Boston surgeon Dario Fauza, MD, a Principal Investigator at Childrens and an affiliate member of the Stem Cell Program, has been investigating the idea of isolating mesenchymal stem cells and using them to grow new tissues for babies who have birth defects detected while they are still in the womb, such as congenital diaphragmatic hernia. These tissues would match the baby genetically, so would not be rejected by the immune system, and could be implanted either in utero or after the baby is born.
3) From pluripotent stem cells: Because embryonic stem cells and induced pluripotent cells (iPS cells), which are functionally similar, are able to create all types of cells and tissues, scientists at Childrens and elsewhere hope to use them to produce many different kinds of adult stem cells. Laboratories around the world are testing different chemical and mechanical factors that might prod embryonic stem cells or iPS cells into forming a particular kind of adult stem cell. Adult stem cells made in this fashion would potentially match the patient genetically, eliminating both the problem of tissue rejection and the need for toxic therapies to suppress the immune system.
4) From other adult stem cells: A number of research groups have reported that certain kinds of adult stem cells can transform, or differentiate, into apparently unrelated cell types (such as brain stem cells that differentiate into blood cells or blood-forming cells that differentiate into cardiac muscle cells). This phenomenon, called transdifferentiation, has been reported in some animals. However, its still far from clear how versatile adult stem cells really are, whether transdifferentiation can occur in human cells, or whether it could be made to happen reliably in the lab.
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Where Do We Get Adult Stem Cells? | Boston Children’s Hospital
This article is about the medical therapy. For the cell type, see Stem cell.
Stem-cell therapy is the use of stem cells to treat or prevent a disease or condition.
Bone marrow transplant is the most widely used stem-cell therapy, but some therapies derived from umbilical cord blood are also in use. Research is underway to develop various sources for stem cells, and to apply stem-cell treatments for neurodegenerative diseases and conditions such as diabetes, heart disease, and other conditions.
Stem-cell therapy has become controversial following developments such as the ability of scientists to isolate and culture embryonic stem cells, to create stem cells using somatic cell nuclear transfer and their use of techniques to create induced pluripotent stem cells. This controversy is often related to abortion politics and to human cloning. Additionally, efforts to market treatments based on transplant of stored umbilical cord blood have been controversial.
For over 30 years, bone marrow has been used to treat cancer patients with conditions such as leukaemia and lymphoma; this is the only form of stem-cell therapy that is widely practiced. During chemotherapy, most growing cells are killed by the cytotoxic agents. These agents, however, cannot discriminate between the leukaemia or neoplastic cells, and the hematopoietic stem cells within the bone marrow. It is this side effect of conventional chemotherapy strategies that the stem-cell transplant attempts to reverse; a donor’s healthy bone marrow reintroduces functional stem cells to replace the cells lost in the host’s body during treatment. The transplanted cells also generate an immune response that helps to kill off the cancer cells; this process can go too far, however, leading to graft vs host disease, the most serious side effect of this treatment.
Another stem-cell therapy called Prochymal, was conditionally approved in Canada in 2012 for the management of acute graft-vs-host disease in children who are unresponsive to steroids. It is an allogenic stem therapy based on mesenchymal stem cells (MSCs) derived from the bone marrow of adult donors. MSCs are purified from the marrow, cultured and packaged, with up to 10,000 doses derived from a single donor. The doses are stored frozen until needed.
The FDA has approved five hematopoietic stem-cell products derived from umbilical cord blood, for the treatment of blood and immunological diseases.
In 2014, the European Medicines Agency recommended approval of Holoclar, a treatment involving stem cells, for use in the European Union. Holoclar is used for people with severe limbal stem cell deficiency due to burns in the eye.
In March 2016 GlaxoSmithKline’s Strimvelis (GSK2696273) therapy for the treatment ADA-SCID was recommended for EU approval.
Stem cells are being studied for a number of reasons. The molecules and exosomes released from stem cells are also being studied in a effort to make medications.
Research has been conducted on the effects of stem cells on animal models of brain degeneration, such as in Parkinson’s, Amyotrophic lateral sclerosis, and Alzheimer’s disease. There have been preliminary studies related to multiple sclerosis.
Healthy adult brains contain neural stem cells which divide to maintain general stem-cell numbers, or become progenitor cells. In healthy adult laboratory animals, progenitor cells migrate within the brain and function primarily to maintain neuron populations for olfaction (the sense of smell). Pharmacological activation of endogenous neural stem cells has been reported to induce neuroprotection and behavioral recovery in adult rat models of neurological disorder.
Stroke and traumatic brain injury lead to cell death, characterized by a loss of neurons and oligodendrocytes within the brain. A small clinical trial was underway in Scotland in 2013, in which stem cells were injected into the brains of stroke patients.
Clinical and animal studies have been conducted into the use of stem cells in cases of spinal cord injury.
The pioneering work by Bodo-Eckehard Strauer has now been discredited by the identification of hundreds of factual contradictions. Among several clinical trials that have reported that adult stem-cell therapy is safe and effective, powerful effects have been reported from only a few laboratories, but this has covered old and recent infarcts as well as heart failure not arising from myocardial infarction. While initial animal studies demonstrated remarkable therapeutic effects, later clinical trials achieved only modest, though statistically significant, improvements. Possible reasons for this discrepancy are patient age, timing of treatment and the recent occurrence of a myocardial infarction. It appears that these obstacles may be overcome by additional treatments which increase the effectiveness of the treatment or by optimizing the methodology although these too can be controversial. Current studies vary greatly in cell-procuring techniques, cell types, cell-administration timing and procedures, and studied parameters, making it very difficult to make comparisons. Comparative studies are therefore currently needed.
Stem-cell therapy for treatment of myocardial infarction usually makes use of autologous bone-marrow stem cells (a specific type or all), however other types of adult stem cells may be used, such as adipose-derived stem cells. Adult stem cell therapy for treating heart disease was commercially available in at least five continents as of 2007.
Possible mechanisms of recovery include:
It may be possible to have adult bone-marrow cells differentiate into heart muscle cells.
The first successful integration of human embryonic stem cell derived cardiomyocytes in guinea pigs (mouse hearts beat too fast) was reported in August 2012. The contraction strength was measured four weeks after the guinea pigs underwent simulated heart attacks and cell treatment. The cells contracted synchronously with the existing cells, but it is unknown if the positive results were produced mainly from paracrine as opposed to direct electromechanical effects from the human cells. Future work will focus on how to get the cells to engraft more strongly around the scar tissue. Whether treatments from embryonic or adult bone marrow stem cells will prove more effective remains to be seen.
In 2013 the pioneering reports of powerful beneficial effects of autologous bone marrow stem cells on ventricular function were found to contain “hundreds” of discrepancies. Critics report that of 48 reports there seemed to be just five underlying trials, and that in many cases whether they were randomized or merely observational accepter-versus-rejecter, was contradictory between reports of the same trial. One pair of reports of identical baseline characteristics and final results, was presented in two publications as, respectively, a 578 patient randomized trial and as a 391 patient observational study. Other reports required (impossible) negative standard deviations in subsets of patients, or contained fractional patients, negative NYHA classes. Overall there were many more patients published as having receiving stem cells in trials, than the number of stem cells processed in the hospital’s laboratory during that time. A university investigation, closed in 2012 without reporting, was reopened in July 2013.
One of the most promising benefits of stem cell therapy is the potential for cardiac tissue regeneration to reverse the tissue loss underlying the development of heart failure after cardiac injury.
Initially, the observed improvements were attributed to a transdifferentiation of BM-MSCs into cardiomyocyte-like cells. Given the apparent inadequacy of unmodified stem cells for heart tissue regeneration, a more promising modern technique involves treating these cells to create cardiac progenitor cells before implantation to the injured area.
The specificity of the human immune-cell repertoire is what allows the human body to defend itself from rapidly adapting antigens. However, the immune system is vulnerable to degradation upon the pathogenesis of disease, and because of the critical role that it plays in overall defense, its degradation is often fatal to the organism as a whole. Diseases of hematopoietic cells are diagnosed and classified via a subspecialty of pathology known as hematopathology. The specificity of the immune cells is what allows recognition of foreign antigens, causing further challenges in the treatment of immune disease. Identical matches between donor and recipient must be made for successful transplantation treatments, but matches are uncommon, even between first-degree relatives. Research using both hematopoietic adult stem cells and embryonic stem cells has provided insight into the possible mechanisms and methods of treatment for many of these ailments.
Fully mature human red blood cells may be generated ex vivo by hematopoietic stem cells (HSCs), which are precursors of red blood cells. In this process, HSCs are grown together with stromal cells, creating an environment that mimics the conditions of bone marrow, the natural site of red-blood-cell growth. Erythropoietin, a growth factor, is added, coaxing the stem cells to complete terminal differentiation into red blood cells. Further research into this technique should have potential benefits to gene therapy, blood transfusion, and topical medicine.
In 2004, scientists at King’s College London discovered a way to cultivate a complete tooth in mice and were able to grow bioengineered teeth stand-alone in the laboratory. Researchers are confident that the tooth regeneration technology can be used to grow live teeth in human patients.
In theory, stem cells taken from the patient could be coaxed in the lab turning into a tooth bud which, when implanted in the gums, will give rise to a new tooth, and would be expected to be grown in a time over three weeks. It will fuse with the jawbone and release chemicals that encourage nerves and blood vessels to connect with it. The process is similar to what happens when humans grow their original adult teeth. Many challenges remain, however, before stem cells could be a choice for the replacement of missing teeth in the future.
Research is ongoing in different fields, alligators which are polyphyodonts grow up to 50 times a successional tooth (a small replacement tooth) under each mature functional tooth for replacement once a year.
Heller has reported success in re-growing cochlea hair cells with the use of embryonic stem cells.
Since 2003, researchers have successfully transplanted corneal stem cells into damaged eyes to restore vision. “Sheets of retinal cells used by the team are harvested from aborted fetuses, which some people find objectionable.” When these sheets are transplanted over the damaged cornea, the stem cells stimulate renewed repair, eventually restore vision. The latest such development was in June 2005, when researchers at the Queen Victoria Hospital of Sussex, England were able to restore the sight of forty patients using the same technique. The group, led by Sheraz Daya, was able to successfully use adult stem cells obtained from the patient, a relative, or even a cadaver. Further rounds of trials are ongoing.
In April 2005, doctors in the UK transplanted corneal stem cells from an organ donor to the cornea of Deborah Catlyn, a woman who was blinded in one eye when acid was thrown in her eye at a nightclub. The cornea, which is the transparent window of the eye, is a particularly suitable site for transplants. In fact, the first successful human transplant was a cornea transplant. The absence of blood vessels within the cornea makes this area a relatively easy target for transplantation. The majority of corneal transplants carried out today are due to a degenerative disease called keratoconus.
The University Hospital of New Jersey reports that the success rate for growth of new cells from transplanted stem cells varies from 25 percent to 70 percent.
In 2014, researchers demonstrated that stem cells collected as biopsies from donor human corneas can prevent scar formation without provoking a rejection response in mice with corneal damage.
In January 2012, The Lancet published a paper by Steven Schwartz, at UCLA’s Jules Stein Eye Institute, reporting two women who had gone legally blind from macular degeneration had dramatic improvements in their vision after retinal injections of human embryonic stem cells.
In June 2015, the Stem Cell Ophthalmology Treatment Study (SCOTS), the largest adult stem cell study in ophthalmology ( http://www.clinicaltrials.gov NCT # 01920867) published initial results on a patient with optic nerve disease who improved from 20/2000 to 20/40 following treatment with bone marrow derived stem cells.
Diabetes patients lose the function of insulin-producing beta cells within the pancreas. In recent experiments, scientists have been able to coax embryonic stem cell to turn into beta cells in the lab. In theory if the beta cell is transplanted successfully, they will be able to replace malfunctioning ones in a diabetic patient.
Human embryonic stem cells may be grown in cell culture and stimulated to form insulin-producing cells that can be transplanted into the patient.
However, clinical success is highly dependent on the development of the following procedures:
Clinical case reports in the treatment orthopaedic conditions have been reported. To date, the focus in the literature for musculoskeletal care appears to be on mesenchymal stem cells. Centeno et al. have published MRI evidence of increased cartilage and meniscus volume in individual human subjects. The results of trials that include a large number of subjects, are yet to be published. However, a published safety study conducted in a group of 227 patients over a 3-4-year period shows adequate safety and minimal complications associated with mesenchymal cell transplantation.
Wakitani has also published a small case series of nine defects in five knees involving surgical transplantation of mesenchymal stem cells with coverage of the treated chondral defects.
Stem cells can also be used to stimulate the growth of human tissues. In an adult, wounded tissue is most often replaced by scar tissue, which is characterized in the skin by disorganized collagen structure, loss of hair follicles and irregular vascular structure. In the case of wounded fetal tissue, however, wounded tissue is replaced with normal tissue through the activity of stem cells. A possible method for tissue regeneration in adults is to place adult stem cell “seeds” inside a tissue bed “soil” in a wound bed and allow the stem cells to stimulate differentiation in the tissue bed cells. This method elicits a regenerative response more similar to fetal wound-healing than adult scar tissue formation. Researchers are still investigating different aspects of the “soil” tissue that are conducive to regeneration.
Culture of human embryonic stem cells in mitotically inactivated porcine ovarian fibroblasts (POF) causes differentiation into germ cells (precursor cells of oocytes and spermatozoa), as evidenced by gene expression analysis.
Human embryonic stem cells have been stimulated to form Spermatozoon-like cells, yet still slightly damaged or malformed. It could potentially treat azoospermia.
In 2012, oogonial stem cells were isolated from adult mouse and human ovaries and demonstrated to be capable of forming mature oocytes. These cells have the potential to treat infertility.
Destruction of the immune system by the HIV is driven by the loss of CD4+ T cells in the peripheral blood and lymphoid tissues. Viral entry into CD4+ cells is mediated by the interaction with a cellular chemokine receptor, the most common of which are CCR5 and CXCR4. Because subsequent viral replication requires cellular gene expression processes, activated CD4+ cells are the primary targets of productive HIV infection. Recently scientists have been investigating an alternative approach to treating HIV-1/AIDS, based on the creation of a disease-resistant immune system through transplantation of autologous, gene-modified (HIV-1-resistant) hematopoietic stem and progenitor cells (GM-HSPC).
On 23 January 2009, the US Food and Drug Administration gave clearance to Geron Corporation for the initiation of the first clinical trial of an embryonic stem-cell-based therapy on humans. The trial aimed evaluate the drug GRNOPC1, embryonic stem cell-derived oligodendrocyte progenitor cells, on patients with acute spinal cord injury. The trial was discontinued in November 2011 so that the company could focus on therapies in the “current environment of capital scarcity and uncertain economic conditions”. In 2013 biotechnology and regenerative medicine company BioTime (NYSEMKT:BTX) acquired Geron’s stem cell assets in a stock transaction, with the aim of restarting the clinical trial.
Scientists have reported that MSCs when transfused immediately within few hours post thawing may show reduced function or show decreased efficacy in treating diseases as compared to those MSCs which are in log phase of cell growth(fresh), so cryopreserved MSCs should be brought back into log phase of cell growth in invitro culture before these are administered for clinical trials or experimental therapies, re-culturing of MSCs will help in recovering from the shock the cells get during freezing and thawing. Various clinical trials on MSCs have failed which used cryopreserved product immediately post thaw as compared to those clinical trials which used fresh MSCs.
There is widespread controversy over the use of human embryonic stem cells. This controversy primarily targets the techniques used to derive new embryonic stem cell lines, which often requires the destruction of the blastocyst. Opposition to the use of human embryonic stem cells in research is often based on philosophical, moral, or religious objections. There is other stem cell research that does not involve the destruction of a human embryo, and such research involves adult stem cells, amniotic stem cells, and induced pluripotent stem cells.
Stem-cell research and treatment was practiced in the People’s Republic of China. The Ministry of Health of the People’s Republic of China has permitted the use of stem-cell therapy for conditions beyond those approved of in Western countries. The Western World has scrutinized China for its failed attempts to meet international documentation standards of these trials and procedures.
State-funded companies based in the Shenzhen Hi-Tech Industrial Zone treat the symptoms of numerous disorders with adult stem-cell therapy. Development companies are currently focused on the treatment of neurodegenerative and cardiovascular disorders. The most radical successes of Chinese adult stem cell therapy have been in treating the brain. These therapies administer stem cells directly to the brain of patients with cerebral palsy, Alzheimer’s, and brain injuries.
Since 2008 many universities, centers and doctors tried a diversity of methods; in Lebanon proliferation for stem cell therapy, in-vivo and in-vitro techniques were used, Thus this country is considered the launching place of the Regentime procedure. http://www.researchgate.net/publication/281712114_Treatment_of_Long_Standing_Multiple_Sclerosis_with_Regentime_Stem_Cell_Technique The regenerative medicine also took place in Jordan and Egypt.
Stem-cell treatment is currently being practiced at a clinical level in Mexico. An International Health Department Permit (COFEPRIS) is required. Authorized centers are found in Tijuana, Guadalajara and Cancun. Currently undergoing the approval process is Los Cabos. This permit allows the use of stem cell.
In 2005, South Korean scientists claimed to have generated stem cells that were tailored to match the recipient. Each of the 11 new stem cell lines was developed using somatic cell nuclear transfer (SCNT) technology. The resultant cells were thought to match the genetic material of the recipient, thus suggesting minimal to no cell rejection.
As of 2013, Thailand still considers Hematopoietic stem cell transplants as experimental. Kampon Sriwatanakul began with a clinical trial in October 2013 with 20 patients. 10 are going to receive stem-cell therapy for Type-2 diabetes and the other 10 will receive stem-cell therapy for emphysema. Chotinantakul’s research is on Hematopoietic cells and their role for the hematopoietic system function in homeostasis and immune response.
Today, Ukraine is permitted to perform clinical trials of stem-cell treatments (Order of the MH of Ukraine 630 “About carrying out clinical trials of stem cells”, 2008) for the treatment of these pathologies: pancreatic necrosis, cirrhosis, hepatitis, burn disease, diabetes, multiple sclerosis, critical lower limb ischemia. The first medical institution granted the right to conduct clinical trials became the “Institute of Cell Therapy”(Kiev).
Other countries where doctors did stem cells research, trials, manipulation, storage, therapy: Brazil, Cyprus, Germany, Italy, Israel, Japan, Pakistan, Philippines, Russia, Switzerland, Turkey, United Kingdom, India, and many others.
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Stem-cell therapy – Wikipedia, the free encyclopedia
Although accumulating data support the efficacy of intramyocardial cell-based therapy to improve left ventricular (LV) function in patients with chronic ischemic cardiomyopathy undergoing CABG, the underlying mechanism and impact of cell injection site remain controversial. Mesenchymal stem cells (MSCs) improve LV structure and function through several effects including reducing fibrosis, neoangiogenesis, and neomyogenesis.
To test the hypothesis that the impact on cardiac structure and function after intramyocardial injections of autologous MSCs results from a concordance of prorecovery phenotypic effects.
Six patients were injected with autologous MSCs into akinetic/hypokinetic myocardial territories not receiving bypass graft for clinical reasons. MRI was used to measure scar, perfusion, wall thickness, and contractility at baseline, at 3, 6, and 18 months and to compare structural and functional recovery in regions that received MSC injections alone, revascularization alone, or neither. A composite score of MRI variables was used to assess concordance of antifibrotic effects, perfusion, and contraction at different regions. After 18 months, subjects receiving MSCs exhibited increased LV ejection fraction (+9.4 1.7%, P=0.0002) and decreased scar mass (-47.5 8.1%; P
Intramyocardial injection of autologous MSCs into akinetic yet nonrevascularized segments produces comprehensive regional functional restitution, which in turn drives improvement in global LV function. These findings, although inconclusive because of lack of placebo group, have important therapeutic and mechanistic hypothesis-generating implications.
http://clinicaltrials.gov/show/NCT00587990. Unique identifier: NCT00587990.
The regenerative potential of the heart is insufficient to fully restore functioning myocardium after injury, motivating the quest for a cell-based replacement strategy. Bone marrow-derived mesenchymal stem cells (MSCs) have the capacity for cardiac repair that appears to exceed their capacity for differentiation into cardiac myocytes.
Here, we test the hypothesis that bone marrow derived MSCs stimulate the proliferation and differentiation of endogenous cardiac stem cells (CSCs) as part of their regenerative repertoire.
Female Yorkshire pigs (n=31) underwent experimental myocardial infarction (MI), and 3 days later, received transendocardial injections of allogeneic male bone marrow-derived MSCs, MSC concentrated conditioned medium (CCM), or placebo (Plasmalyte). A no-injection control group was also studied. MSCs engrafted and differentiated into cardiomyocytes and vascular structures. In addition, endogenous c-kit(+) CSCs increased 20-fold in MSC-treated animals versus controls (P
MSCs stimulate host CSCs, a new mechanism of action underlying successful cell-based therapeutics.
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Bone marrow mesenchymal stem cells stimulate cardiac stem …
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16.01.2015 – Press release
Circulation, metabolism, nutrition
On the 21 October 2014, Professor Philippe Menasch and his team from the cardiovascular surgery service of the Georges Pompidou European Hospital, AP-HP, carried out a transplant of cardiac cells derived from human embryonic stem cells*, according to a method developed by the Department of Cell and Tissue Biotherapies of the Saint-Louis hospital, directed by Professor Jrme Larghero and through research led by this group within Inserm. The surgery, coupled with a coronary bypass*, was carried out on a woman of 68 years suffering from severe heart failure. Ten weeks after the intervention, the patient is feeling well, her condition has improved markedly, with no complications having been observed. This promising advance was presented this Friday, 16 January 2015 at the XXV European Days Conference of the French Society of Cardiology.
Human embryonic stem cells. Transplantation of undifferentiated human embryonic stem cells into rat heart organotypic cultures. Presence of human cells, in the cardiac parenchyma of the rat two months after injection. The human cells are positive for human nuclear antigen marking (red). Cardiac rat tissue is positive for cardiac troponin 1 marking (green). I-Stem (Institiute for Stem Cell Therapy), Evry Genopole. Inserm/Habeler, Walter
The transplant was carried out as part of a clinical trial developed by the Public Hospitals of Paris (AP-HP) and through the work of the teams from AP-HP, Inserm and the universities of Paris-Descartes and Paris-Diderot. The cardiac cells were prepared according to a technique developed by the Department of Cell and Tissue Biotherapies of the Saint-Louis hospital. The cytogenetics laboratory of the Antoine Bclre Hospital and the French General Agency for Health Products and Equipment also contributed to the preparation of this phase I trial which will enable the verification of the safety and feasibility of the procedure
For 20 years Professor Menasch, currently co-director of an Inserm team within PARCC (Paris Centre for Cardiovascular Research), and his colleagues have been involved in stem cell* therapy for heart failure.
The team first tested the implant of skeletal muscle stem cells in necrosed areas of the heart in the laboratory. These cells were implanted into the heart of a patient with heart failure for the first time in the world on 15 June 2000. Following an initial series of these implants, always coupled with a coronary bypass, the team coordinated a European multi-centre, randomised, placebo-controlled trial whose results have still not been able to establish any significant benefit of these cells on the contractile function of patients hearts. One of the conclusions drawn from this trial was that to be fully efficient, transplanted cells should resemble the cells of the tissue to be repaired as much as possible, in this instance cardiac tissue. It was then decided to venture along the path of embryonic stem cells. Derived from embryos conceived in in vitro fertilisation, these cells do in fact possess pluripotent properties, that is, they are capable of developing into any type of cell of the body, including of course cardiac cells, as soon as they receive the appropriate signals during the culture cycle in the laboratory.
In 2007, the team then composed of, among others, Michel Pucat, Director of Research at Inserm, and Philippe Menasch showed that human embryonic stem cells could be differentiated into cardiac cells after being transplanted into the failing hearts of rats. Since then, many experiments have been carried out on different animal species in order to validate the efficacity of these cells and to optimise conditions which can guarantee maximum safety. At the end of this stage, a bank of pluripotent embryonic stem cells was formed in the conditions which satisfied all regulatory constraints applying to biological products for human use. Then, the Department of Cell and Tissue Biotherapies of the Saint-Louis hospital, still in liaison with the Inserm teams, developed and tested specialisation procedures for cells in order to produce young cardiac cells from them. The focus was then on the purification of the cells directed like so in order to ensure that the final product was expunged of any remaining pluripotent cells which could be potentially tumorigenic.
Besides, as initial experience with muscular stem cells showed the limitations of administering cells by multiple injections, their transfer is now performed using a patch that the cells are incorporated into. This patch is then placed on the area of the infarction. To that end, after the purification stage, the cardiac cells are incorporated into a circular fibrin gel which is applied, during the surgical procedure, to the necrosed area with just a few sutures ensuring that it is anchored to the cardiac tissue.
This type of surgery is aimed at serious heart failure which doesnt respond to the usual medicinal treatments but is not at the stage of a complete heart transplant. This is a promising advance, which we hope will enrich the therapeutic arsenal available to treat heart failure today explains Prof. Menasch. We are continuing the trial, which authorises us to carry out four other transplants. It would seem already that the benefits of the cells are linked mainly to the substances that they secrete. The direct administration of the substances, without going through a transplant of productive cells, is a path to explore.
This project has been entirely financed by funds from public intstitutions and societies and was authorised by the French National Agency for the Safety of Medicines and Health Products (ANSM) after agreement with the Agency for Biomedicine for the importation and research on human embryonic cells.
Cell therapy: refers to cell transplants aiming to restore the function of tissue or an organ when it has been altered by an accident, illness or ageing. These therapies have benefited from recent scientific advances on stem cells and give millions of patients the hope of regenerative medicine.
Embryonic or pluripotent stem cells: they can renew indefinitely (self-renewal), multiply in a culture and be differentiated into more than 200 types of cell. In the course of development, they are destined to form all types of the bodys tissue.
Coronary bypass: a technique that enables the redirection of the bloodstream towards the cardiac muscle, by using a graft (coming from the saphenous vein or from a thoracic artery.) One end of the graft is connected to the aorta, the main artery supplying the coronary arteries; the other end is connected to the coronary artery, situated just behind the site of the obstruction. This creates a detour enabling the oxygenated blood to circulate towards the heart.
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Stem cell therapy for heart failure: first implant of …
Stem cell therapies for post-heart attack tissue repair have had modest success at best. Clinical trials have primarily used bone marrow cells, which can promote the growth of new blood vessels, but many studies have shown no benefit. A better alternative may be to use human heart muscle cells (cardiomyocytes), suggests a study published October 22 in Stem Cell Reports, the journal of the International Society for Stem Cell Research.
The authors compared how well human embryonic stem cell-derived cardiomyocytes, embryonic stem cell-derived cardiovascular progenitors, and bone marrow cells worked to repair tissue damage post-heart attack in a rat. The verdict is that both cardiomyocytes and progenitor cells surpassed the healing power of bone marrow cells. And despite the progenitors’ abilities to differentiate into more cell types, they demonstrated no statistically significant improvement in heart tissue function, which means the more mature and stable heart muscle cells are a viable option for future therapies.
“There’s no reason to go back to more primitive cells, because they don’t seem to have a practical advantage over more definitive cell types in which the risk for tumor formation is lower,” says senior study author Charles Murry of the University of Washington, Seattle. “The other important finding is that both of these populations are far superior to bone marrow cells. This work is a go signal that tells us to keep moving on to more promising and more powerful cell types in human trials.”
The experiments, led by first authors Sarah Fernandes and James J.H. Chong, involved injecting the cells in the walls of the heart and measuring how well heart muscle tissue contracted in follow-up tests 4 weeks later. About ten animals receiving each of the three treatment variables and ten controls receiving a non-therapeutic cell population were included in the study. Injections were given 4 days after heart attacks occurred in the rats, as interventions that are given later don’t have as much impact.
James Chong, now an interventional cardiologist at the University of Sydney, added: “We have recently had success in regenerating hearts of monkeys using a similar approach of transplanting stem cell-derived cardiomyocytes. The next goals will be to determine if these large animal experiments show similar improvements in cardiac function, and if so, to begin testing these cells in human patients.”
The above post is reprinted from materials provided by Cell Press. Note: Materials may be edited for content and length.
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Cardiac muscle cells as good as progenitors for heart …
c-kit is expressed in the developing and adult mouse heart
We first generated a knock-in mouse model, c-kitH2B-tdTomato/+, by gene targeting (Fig. 1a and Supplementary Fig. 1). In this animal, the H2B-tdTomato cassette was inserted into the c-kit start codon without deleting any genomic sequences, thereby expressing tdTomato under the control of the full complement of endogenous c-kit regulatory elements. Since tdTomato is fused to histone H2B gene24, its expression is localized to the nucleus.
(a) Diagram of the c-kitH2B-tdTomato/+knock-in allele. (bi) Sections of c-kitH2B-tdTomato/+hearts at embryonic days (E) 8.5, 9.5, 12.5 and 14.5 (be) and at postnatal (P) days 1, 30, 60 and 120 (fi). c2, e2, g2 and i2 are high-magnification images (without DAPI) of the areas outlined in c1, e1, g1 and i1, respectively. c-kitH2B-tdTomato cells are denoted by arrows. LA, left atria; LV, left ventricle; OFT, outflow tract; RA, right atria; RV, right ventricle; VS, ventricular septum. n=3 for each stage. Scale bar, 100m.
To confirm the fidelity of the c-kitH2B-tdTomato signal to the endogenous c-kit expression pattern, we performed whole-mount RNA in situ hybridization on the wild-type mice from embryonic day (E) 9.5 to E13.5. By comparing c-kitH2B-tdTomato signals to c-kit mRNA expression, we found that the signals overlapped in all known regions of c-kit expression25, 26, such as the pharyngeal arches, liver, umbilical cord and melanocytes (Supplementary Fig. 2ac). Furthermore, H2B-tdTomato expression was detected in other organs, including the lung, stomach, intestine and spleen (Supplementary Fig. 2e), as well as the neural tube and yolk sac during embryogenesis. This finding is consistent with previous reports of c-kit expression in these organs25, 26. Immunostaining of sectioned c-kitH2B-tdTomato/+ mouse tissues revealed that the c-kitH2B-tdTomato-positive cells co-localized with c-kit antibody in the liver, lung and melanocytes (Supplementary Fig. 3). Further support for the sensitivity and fidelity of this reporter is the observation that cells with low c-kit expression detected by antibody exhibited bright H2B-tdTomato fluorescence (Supplementary Fig. 3b,c).
Next, we examined the location of c-kit+ cells in the hearts of c-kitH2B-tdTomato/+mice (Fig. 1). Endocardial cells with nuclear tdTomato expression were observed as early as E8.5 and 9.5 (Fig. 1b,c). Starting from E12.5, cells with strong c-kitH2B-tdTomato expression were dispersed throughout the heart, with the highest density in the inner layers of the atrial and ventricular chambers at all embryonic stages tested (Fig. 1d,e). At postnatal day (P) 1, P30, P60 and P120, c-kitH2B-tdTomatoexpressing cells were consistently detected in all chambers of the heart (Fig. 1fi). The broad distribution of c-kitH2B-tdTomato-positive cells in the heart from embryonic stages to adulthood is inconsistent with previous studies reporting that c-kit+ cells represent a small population of CSCs in the mammalian heart7, 12, 13, 14, 15, 27.
In the initial characterization of cardiac resident c-kit+ cells in the adult rat, c-kit+ cells were shown to contain a mixed population of cells exhibiting early stages of myogenic differentiation as demonstrated by the active expression of the early cardiac transcription factors Nkx2.5, Gata4 and Mef2c in the nucleus and of sarcomeric proteins in the cytoplasm of these cells7, 15. To determine whether c-kitH2B-tdTomato-positive cells express the cardiac progenitor marker Nkx2.5, we crossed Nkx2.5H2B-GFP/+knock-in mice28 with c-kitH2B-tdTomato/+mice to obtain compound heterozygotes (c-kitH2B-tdTomato/+;Nkx2.5H2B-GFP/+). H2BGFP expression in Nkx2.5H2B-GFP/+mice faithfully recapitulates the endogenous Nkx2.5 pattern28. We examined cardiac tissues throughout the embryonic (E9.518.5) and postnatal (P1120) stages (Supplementary Fig. 4). All histological sections from E9.5 to 13.5 hearts and more than 30 sections from E14.5 to P120 hearts were inspected (n=3 for each stage). However, no c-kitH2B-tdTomato and Nkx2.5H2B-GFP double-positive cells were found (Supplementary Fig. 4b,dg), except at E12.5, wherein only 11 double-positive cells were detected in the ventricular septum (Supplementary Fig. 4c, ~0.007% of total Nkx2.5H2B-GFP-positive cells).
To determine whether any c-kit+ cells produce sarcomeric or myocardial proteins7, 15, we applied a cTnTH2B-GFP/+ knock-in mouse model with insertion of an H2BGFP cassette into the start codon of cTnT (Tnnt2; Supplementary Fig. 5a). On examining heart sections from c-kitH2B-tdTomato/+;cTnTH2B-GFP/+ compound heterozygous animals at embryonic and postnatal stages (E8.5P120), we did not detect any cells in which both markers were co-localized (Supplementary Fig. 5), with the exception of E13.5, where an average of 15 double-positive cells were found within the ventricular septum (Supplementary Fig. 5d, ~0.009% of total cTnTH2B-GFP-positive cells). These observations reveal that c-kit+ cells in c-kitH2B-tdTomato/+mice very rarely co-express either Nkx2.5 or cTnT in the embryonic heart and do not co-express these markers in foetal or adult hearts.
To further determine the identity of c-kit+ cells, we performed immunostaining with antibodies against the endothelial marker PECAM (CD31) and the smooth muscle marker, -SMA. Surprisingly, at all the stages examined (E8.5P120), c-kitH2B-tdTomato-positive cells were PECAM+(Fig. 2a-f) but -SMA (Fig. 2g,h). This finding suggests that cardiac c-kitH2B-tdTomato-positive cells are endothelial cells. Quantitative flow cytometric analysis of 4-month-old hearts demonstrated that ~43% PECAM+ cells in the ventricles were also c-kit+ (Supplementary Fig. 6). Thus, our results indicate that c-kitH2B-tdTomato-positive cells represent a subset of cardiac endothelial cells.
(a,b) At E8.5 and E9.5, c-kitH2B-tdTomato cells are endocardial (PECAM+). (cf) c-kitH2B-tdTomato cells express PECAM at E16.5 (c) and at P1120 (df). Arrows indicate PECAM+ and tdTomato+ double-positive cells. Arrowheads indicate PECAM+ and tdTomato cells. (g,h) Cardiac smooth muscle cells (-SMA+) are tdTomato at P120 (arrowheads). a2h2 are high-magnification images of the areas outlined in a1h1 (without DAPI), respectively. n=3 for each stage. Scale bar, 100m.
tdTomato is a bright fluorescent protein29, 30. We were concerned that the long stability of tdTomato could complicate the detection of transient c-kit expression. To confirm the identity of c-kit+ cells identified by c-kitH2B-tdTomato/+, we generated another reporter line, c-kitnlacZ-H2B-GFP/+, by inserting a LoxP-nlacZ-4XPolyA-LoxP-H2BGFP cassette into the c-kit start codon (Fig. 3a and Supplementary Fig. 7). H2BGFP is not detected in this line unless the nlacZ-4XPolyA stop cassette is removed by Cre-mediated recombination. We performed whole-mount X-gal staining on c-kitnlacZ-H2B-GFP/+ embryos and found that the c-kitnlacZ signal was not only reliably recapitulated by c-kit mRNA expression, but also consistent with the H2BtdTomato expression patterns in c-kitH2B-tdTomato/+mice (Supplementary Fig. 2). Furthermore, X-gal staining of whole-mount and sectioned hearts at E15.5P90 readily detected a broad distribution of c-kitnlacZ-positive cells throughout the heart (Fig. 3b,d,f,h, and j), including the endocardium (Fig. 3b,h), similar to the pattern observed in c-kitH2B-tdTomato/+mice. X-gal staining of compound heterozygous littermate hearts bearing an endothelial-specific Tie2-Cre allele (c-kitnlacZ-H2B-GFP/+;Tie2Cre) could not detect c-kitnlacZ-positive cells (Fig. 3c,e,g,i and k; less than 10 randomly distributed c-kitnlacZ-positive cells were found in the adult heart, representing ~0.0002% of total c-kit+ cells). Consistent with the endothelial nature of c-kit+ cells in the heart, c-kitH2B-GFP-positive cells generated by Tie2Cre excision were all co-stained with anti-PECAM antibody (Supplementary Fig. 8). Thus, the c-kitnlacZ-H2B-GFP/+ reporter line confirms the endothelial identity of cardiac c-kit+ cells.
(a) Diagram of the c-kitnlacZ-H2B-GFP/+reporter allele (a1). The c-kitH2B-GFP/+ allele is generated when the nlacZ cassette is removed by Cre excision (a2). (bk) X-gal staining of c-kitnlacZ-H2B-GFP/+ and c-kitnlacZ-H2B-GFP/+;Tie2Cre littermate hearts at E15.5 (b,c, sections) and at P190 (dk). Arrows indicate comparable regions to X-gal+ or X-gal staining. Arrowheads indicate rare X-gal+ cells on c-kitnlacZ-H2B-GFP/+;Tie2Cre hearts, suggesting that most c-kit+ cells lose the nlacZ gene because they are in the Tie2Cre lineage. f2k2 are high-magnification images of the areas outlined in f1k1, respectively. n=35 for each stage. Scale bar, 400m (black) and 200m (white).
To further address the issue of stability of both H2BtdTomato and nlacZ proteins, we analysed cardiac c-kit cells with the third reporter allele c-kitMerCreMer/+, in which an inducible MerCreMer cassette was inserted into the c-kit start codon (Fig. 4a and Supplementary Fig. 9). c-kitMerCreMer/+;ROSA26RtdTomato/+mice were subsequently generated by crossing with ROSA26RtdTomato/+ mice. In the absence of tamoxifen treatment, no tdTomato-expressing cells were detected in the adult hearts. To confirm whether c-kit is actively expressed in the postnatal heart, we injected tamoxifen at P30, P60 or P90 for 3 consecutive days (days 1, 2 and 3), and immediately collected cardiac tissues for analysis at day 4 (P3034, P6064) or 14 (P90104). This treatment consistently resulted in tdTomato labelling of a large number of cells in the heart (Fig. 4b,d,e) that also expressed PECAM (Fig. 4c). This result further confirms that cardiac c-kit+ cells are endothelial (Figs 2 and 3), and supports the previous observation that cardiac c-kit+ cell progeny are endothelial19.
(a) Diagram of the c-kitMerCreMer/+ allele. c-kitMerCreMer/+ animals were crossed to the ROSA26RtdTomato reporter line to obtain c-kitMerCreMer/+;ROSA26RtdTomato/+. (be) Cre activity was transiently induced in c-kitMerCreMer/+;ROSA26RtdTomato/+ animals at P30, P60 and P90 by tamoxifen injection on days 13. Hearts were harvested on days 4 and 14. Many tdTomato+ cells (arrows in b2, d2 and e2) were detected in hearts at P34 (b1), P64 (d1) and P104 (e1). These tdTomato+ cells were PECAM+ (c2, arrows, P3034). b2, d2 and e2 are high-magnification florescent images of the areas outlined in b1, d1 and e1 (bright field), respectively. (f) Diagram of the cTnTnlacZ-H2B-GFP/+allele and lineage tracing using c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+mice. Cre activity was transiently induced by tamoxifen injection for 4 days on days 1, 2, 3 and 5 (days 1 and 2 for E11.5). Samples were collected on day 7 (day 3 for E11.5). (g) cTnTH2B-GFP cells were detected at E13.5, P37, P67 and P97 (arrows), with the total number in the whole heart noted at the upper right corner. Scale bar, 1 mm (black) and 100m (white).
c-kitH2B-tdTomato/+, c-kitnlacZ-H2B-GFP/+ and c-kitMerCreMer/+ animals are heterozygous null for c-kit (c-kit+/). Haploinsufficiency of c-kit could affect c-kit regulation in vivo20, 31, 32, 33, possibly leading to ectopic cardiac expression. To determine whether ectopic c-kit expression occurs in the reporter mouse hearts, we performed immunostaining at embryonic (E11.515.5) and postnatal stages (P160) using c-kit antibody on mice of four different genotypes: wild type, c-kitH2B-tdTomato/+ (c-kit+/), c-kitH2B-tdTomato/MerCreMer(c-kit/) and c-kitMerCreMer/MerCreMer(c-kit/). Using c-kit antibody, we frequently detected cells in wild-type hearts that were dually labelled with c-kit and PECAM (Supplementary Fig. 10a4,d4,g2 and Supplementary Fig. 11a,f,h,i). In c-kitH2B-tdTomato/+ animals, c-kit antibody immunoreactivity co-localized with c-kitH2B-tdTomato (Supplementary Fig. 10b2, e2,h2 and Supplementary Fig. 11b,c), although the immunofluorescence was decreased compared with that in wild-type animals. Reduced c-kit immunoreactivity in c-kitH2B-tdTomato/+ tissues is consistent with the c-kit+/ genetic background (theoretically 50% c-kit protein reduction in c-kit+/). Importantly, c-kit antibody staining was completely undetectable in c-kit/mutant hearts or lungs, even with Tyramide Signal Amplification (TSA) amplification (Supplementary Figs 10c,f and 11d,e), demonstrating the specificity of the antibody staining. Therefore, immunostaining with c-kit antibody also reveals that cardiac c-kit+ cells are endothelial and indicates that no ectopic cardiac c-kit expression occurs in the new knock-in mouse models employed.
To further determine the myogenic potential of c-kit+ cells during heart formation, we applied cTnTnlacZ-H2B-GFP/+ cardiomyocyte-specific reporter mice with the LoxP-nlacZ-4XPolyA-LoxP-H2B-GFP cassette targeted into cTnT start codon. cTnTH2B-GFP expression is detected in cardiomyocytes when Cre is expressed in the myocardium or myogenic precursor cells (Fig. 4f). We crossed c-kitMerCreMer/+ mice with cTnTnlacZ-H2B-GFP/+mice and injected tamoxifen in c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ animals. After two doses of tamoxifen administration (days 1 and 2) to pregnant mice (E11.5 embryos) or four doses (days 1, 2, 3 and 5) to P30, P60 and P90 mice, we collected hearts for analysis at E13.5 or at P37, P67 and P97, respectively. All cardiac sections were assessed for cTnTH2B-GFP-positive cells. On average, approximately 50, 324, 156 and 66 cells were found in hearts (n=3 for each group) at E13.5, P37, P67 and P97, respectively (Fig. 4g), representing
Previous studies have reported that within 4 weeks of myocardial infarction in adult mouse hearts, the number of c-kit/Nkx2.5 double-positive myogenic precursors significantly increased in the injured region, and some of these myogenic precursors transformed into proliferative cardiomyocytes7, 15. To directly investigate the differentiation potential of cardiac c-kit+ cells post myocardial infarction, we ligated the left anterior descending (LAD) coronary artery of c-kitH2B-tdTomato/+;Nkx2.5H2B-GFP/+ mice (25 months old, n=12, Fig. 5a,b). Examination of cardiac sections at 1, 3, 7, 21, 30 and 60 days post-surgery (dps) revealed many c-kitH2B-tdTomato-positive cells in the infarcted region (Fig. 5cf). However, no c-kitH2B-tdTomato and Nkx2.5H2B-GFP double-positive cells were found in the injured area at any stage tested (Fig. 5c1f1). To further determine the cell identity of these c-kit+ cells, we performed LAD ligation on Tie2Cre;c-kitnlacZ-H2B-GFP/+ mice (24 months old, n=3). c-kitH2B-GFP-positive cells were readily detected in the infarcted region, demonstrating that they retained their endothelial nature after injury (Fig. 6a).
(a) Diagram of LAD ligation. (b) Masson trichrome staining shows the infarcted region of a c-kitH2B-tdTomato/+;Nkx2.5H2B-GFP/+heart at 60 days post-surgery (dps). b1 and b2 are high-magnification images of the numbered outlined areas in b. (cf) No c-kitH2B-tdTomato/Nkx2.5H2B-GFP double-positive cells were found in the infarcted regions at 3 (c), 21 (d), 30 (e) and 60dps (f). c1/c2, d1/d2, e1/e2, and f1/f2 are high-magnification images of the numbered outlined areas in c, d, e and f, respectively. Scale bar, 500m (black) and 50m (white).
(a) c-kitH2B-GFP-positive cells were present in the infarcted region of Tie2Cre;c-kitnlacZ-H2B-GFP/+ hearts at 30dps. a2 is green channel of a1, and a3 is high-magnification image of the area outlined in a2. (b) Masson trichrome staining of cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+;ROSA26RtdTomato/+ hearts at 60dps shows the infarcted region. (c) Adjacent section of b. ROSA26RtdTomato signal indicates myocardial cells after tamoxifen induction (c1). No c-kitH2B-GFP cells were observed in the infarcted zone (arrows). c2 is green channel of c1. (d) Masson trichrome staining of c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ hearts at 60dps. (e) Adjacent section of d shows a few cTnTH2B-GFP cells (
A recent study reported that a subpopulation of endothelial cells yields progeny with CSC characteristics in the adult mouse heart34. This subpopulation purportedly arises from endothelialmesenchymal transition and gives rise to cardiomyocytes that contribute to heart renewal34. To determine whether c-kit+ endothelial cells produce CSCs that further differentiate into cardiomyocytes following cardiac injury, we performed LAD ligation on cTnTMerCreMer/+;c-kitnlacZ-H2B-GFP/+;ROSA26RtdTomato/+ mice (24 months old, n=4, Fig. 6b). cTnTMerCreMer/+ mediates specific and effective myocardial recombination after tamoxifen induction35. If c-kitnlacZ-H2B-GFP/+ cells become cardiomyocytes and if c-kit expression is maintained in these cells, then c-kitH2B-GFP-positive cells would be detected. However, after tamoxifen was injected at 37dps and 3135dps (three tamoxifen treatments for each period), we detected no c-kitH2B-GFP-positive cells in the infarcted region (Fig. 6c), although myocardial recombination was widely detected in and adjacent to the infarcted region (as revealed by ROSA26RtdTomato staining, Fig. 6c). Furthermore, examination of adult c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ mice after LAD ligation (35 months old, n=3, Fig. 6d) revealed
In the lineage tracing experiments used to determine the myocardial potential of c-kit+ cells during development and after cardiac injury in c-kitMerCreMer/+;cTnTnlacZ-H2B-GFP/+ animal models, very small number of cTnTH2B-GFP-positive cells was detected (Fig. 4g, ~66156 cells; and Fig. 6e, ~20 cells). In all cases, the number was extremely low when compared with the total number of c-kitH2B-tdTomato-positive cells (
Cardiac myocytes have been traditionally regarded as terminally differentiated cells that adapt to increased work and compensate for disease exclusively through hypertrophy.1 In the past few years, compelling evidence has accumulated suggesting that the heart has regenerative potential.25 The origin and significance of the subpopulation of replicating myocytes are unknown; these issues could be relevant to understand the for mechanisms coaxing endogenous cardiomyocytes to reenter the cell cycle and to the search for strategies to transplant cardiac progenitor cells.6 In fact, although embryonic stem cells have an exceptional capacity for proliferation and differentiation, potential immunogenic, arrhythmogenic, and, particularly, ethical considerations limit their current use. Moreover, autologous transplantation of skeletal myoblasts has been considered because of their high proliferative potential, their commitment to a well-differentiated myogenic lineage, their resistance to ischemia, and their origin, which overcomes ethical, immunological, and availability problems. However, even if phase II clinical trials with autologous skeletal myoblasts are ongoing, several problems related to potentially life-threatening arrhythmia (perhaps reflecting cellular uncoupling with host cardiomyocytes7) must be taken into account when this approach is considered. Furthermore, although cardiomyocytes can be formed, at least ex vivo, from different adult stem cells, the ability of these cells to cross lineage boundaries is currently causing heated debate in the scientific community,8 with the majority of reports indicating neoangiogenesis as the predominant in vivo effect of bone marrow or endothelial progenitor cells.9,10
This report describes the identification and preliminary characterization of cells from the adult human and murine heart, which have the properties of cardiac stem cells. Because these cells also have been isolated and expanded from human heart biopsy specimens, they could have a significant impact on future clinical strategies to treat patients with heart disease.
Human tissue was derived from atrial or ventricular biopsy specimens belonging to patients (1 month to 80 years of age) undergoing heart surgery, in conformation with the guidelines of the Italian Department of Health. Murine tissue was derived from the hearts of previously characterized homozygous MLC1/3F-nlacZ11 and cTnI-nlacZ12 transgenic mice expressing a nuclear lacZ transgene under the transcriptional control of the striated muscle myosin light chain or cTnI promoters, respectively, homozygous B5-eGFP mice,13 homozygous GFP-cKit14 mice, MLC3F-nlacZ/B5-eGFP, MLC3F-nlac-Z/GFP-cKit, and cTnI-nlacZ/B5-eGFP cTnI-nlac-Z/GFP-cKit crossed mice, SCID mice, and SCID beige mice (Charles River Italia, Lecco, Italy).
Isolated myocardial tissue was cut into 1- to 2-mm3 pieces, washed with Ca2+-Mg2+free phosphate-buffered solution (PBS) (Invitrogen), and digested three times for 5 minutes at 37C with 0.2% trypsin (Invitrogen) and 0.1% collagenase IV (Sigma, Milan, Italy). The obtained cells were discarded, and the remaining tissue fragments washed with complete explant medium (CEM) (Iscoves Modified Dulbeccos Medium [IMDM] supplemented with 10% fetal calf serum, 100 U/mL penicillin G, 100 g/mL streptomycin, 2 mmol/L l-glutamine, and 0.1 mmol/L 2-mercaptoethanol) were cultured as explants in CEM at 37C and 5% CO2. After a period ranging from 1 (embryo) to 3 (adult) weeks, a layer of fibroblast-like cells was generated from adherent explants over which small, phase-bright cells migrated. These phase-bright cells were collected by pooling two washes with Ca2+-Mg2+free PBS, one wash with 0.53 mmol/L EDTA (Versene, Invitrogen) (1 to 2 minutes), and one wash with 0.5 g/L trypsin and 0.53 mmol/L EDTA (Invitrogen) (2 to 3 minutes) at room temperature under visual control. The cells obtained (from 104 to 4105 cells/explant) were seeded at 0.5 to 2105 cells/mL in poly-d-lysine-coated multiwell plates (BD Bioscences, Milan, Italy) in cardiosphere-growing medium (CGM) (35% complete IMDM/65% DMEMHam F-12 mix containing 2% B27, 0.1 mmol/L 2-mercaptoethanol, 10 ng/mL epidermal growth factor [EGF], 20 ng/mL basic fibroblast growth factor [bFGF], 40 nmol/L cardiotrophin-1, 40 nmol/L thrombin, antibiotics, and l-Glu, as in CEM). Isolation of the cardiosphere-forming cells could be performed at least 4 times at 6- to 10-day intervals from the same explant. Cardiospheres (CSs) were passaged every 2 to 3 days by partial changing of the medium and mechanical trituration of the larger clusters. Movies of cultured CSs, available in the online data supplement at http://circres.ahajournals.org, were recorded using a Nikon-COOLPIX-4500 digital camera connected to a Leitz inverted microscope. For cryopreservation, we used CEM/DMEMHam F12 at 50:50, 5% B27, and 10% DMSO as the freezing medium.
Extensive descriptions of BrdUrd labeling, clonal analysis, differentiation on substrate-coated surface, coculture experiment, immunocytochemistry, flow cytometric analysis, in vivo analysis, and heterotopic and orthotopic transplantation are provided in the online data supplement.
Sphere-generating cells were obtained by mild enzymatic digestion of explanted human atrial or ventricular biopsy specimens and embryo, fetal, and postnatal mouse hearts. Soon after the generation of a layer of fibroblast-like cells from well-adherent explants, some small, round, phase-bright cells began to migrate over this coat. These cells could be harvested periodically by treatment with EDTA and mild trypsinization and were allowed to grow on poly-d-lysinecoated culture surfaces in a low-serum (3.5% fetal calf serum) medium supplemented with a serum substitute (B27), growth factors (EGF and bFGF), cardiothrophin-1 (CT-1),15 and thrombin.16 During the first week of culture, the last factor led to a 7-fold increase in the number of spheres with respect to that obtained using the medium supplemented with the other factors, either alone or in combination. Time-course observations of cells derived from human and murine explants showed that early after their seeding (30 minutes), some of these cells began to divide while still in suspension. Most cells became loosely adherent, whereas others remained in suspension, and some contaminating fibroblast-like cells attached firmly to the poly-d-lysine coat. Cellular divisions also were evident from the loosely adherent cell population and produced clusters of small, round, phase-bright cells (that we termed CSs) after 10 to 12 hours (Figure 1a). Within 24 to 36 hours of their appearance, CSs increased in size and some of them detached from the culture surface; after 48 to 72 hours, most CSs were between 20 and 150 m in size, and, when not subjected to mechanical dissociation, the largest contained dark zones within the inner mass (Figure 1a).
Figure 1. CS proliferation. a, Phase micrograph of floating CSs (cultured from 48 hours) derived from primary culture of a human atrial biopsy sample. b, Proliferation curves of human and mouse CSs (derived from 8 different subjects [left] and from prenatal and postnatal hearts [middle and right], respectively) in the presence (middle) and absence (right) of 3.5% serum. Number of spheres refers to the mean number per well from which 90% of the spheres were withdrawn at each time point for further analysis. Note the different pattern of proliferation between the human and mouse CSs and the rapid rise of the curves, followed by an irreversible decline in the serum-free conditions.
Murine CSs started beating spontaneously soon after their generation (Supplementary Movie: mouse CSs movie 1a) and maintained this function during their life span (Supplementary Movie: mouse CSs movie 1b), whereas human CSs did so only when cocultured with rat cardiomyocytes (Supplementary Movie: human CSs movie 1a and 1b). To be sure that contraction was a new trait acquired by the CSs cells, GFP-labeled human CSs (partially or totally dissociated) were cocultured with cardiomyocytes prestained (Supplementary Human CSs Movie 2b through 2d) or not prestained (Supplementary Human CSs Movie 3a through 3d) with Dil. Contracting GFP-labeled cells were observed after 48 hours of coculture; furthermore, Cx-43 immunostaining performed on the cocultures of human GFP-transduced CSs with unlabeled neonatal rat cardiomyocytes showed the typical punctuate fluorescence pattern of the main gap junction protein of the heart along the cytoplasmatic membrane of the human cells (Figure 2d and Supplementary Figure VIII), suggesting that a functional connection is created between the two cellular populations.
Figure 2. Clonogenesis and coculture features. a, Fluorescence analysis of a single cell (upper right) (obtained from a dissociated GFP-expressing CS) when plated by limiting dilution on mitomycin-treated STO fibroblast-coated 96-well plates in CGM over the course of the generation of the GFP-labeled clone. This clone could be passaged and expanded on poly-d-lysine coat (lower left). b, X-Gal staining of a eGFP/MLC3F clone (obtained in the same way as were human clones) after 48 hours of exposure to growth factor-free medium. In these conditions, clone cells become more flattened, with many nuclei appearing blue, demonstrating that a differentiation process occurred (see also Supplementary Figure I and Supplementary clone movies). c, Fluorescence analysis of partially dissociated eGFP-labeled human CSs at 96 hours of coculture with rat cardiomyocytes. The same green cells that showed a synchronous contraction with cardiocytes (see supplementary human CSs movies) express cTnI. d, Fluorescent analysis of connexin-43 expression (red) in eGFP-labeled human CSs cocultured with rat cardiomyocytes, as in (c). A punctuate red fluorescence is present in the cell membrane of human cells (see Supplementary Figure VIII).
CSs were found to be composed of clonally derived cells and did not simply represent cellular aggregates. In fact, when human GFP-transduced CSs or murine CSs (derived from eGFP/MLC3F or eGFP/cTrI mice) were dissociated and plated as single cells on mitomycin-treated STO fibroblast-coated 96-well plates (or clonally diluted on 10-cm Petri dishes), fluorescent spheres were generated with a 1% to 10% efficiency (Figure 2a). These spheres could be subcloned on poly-d-lysine-coated surfaces, showing the same functional and phenotypic behavior in culture as the nonclone-derived CSs. In fact, 3 days after their appearance, some of the MLC3F-nlacZ/B5-eGFP or cTnI-nlacZ/B5-eGFP mice clonederived CSs started to beat (supplementary clone movie), and, after 48 hours of culture with CEM, the majority (6 of 7) of these showed expression of the lac-Z transgene within the nuclei after specific histochemical staining (Figure 2b1 and 2b2 and Supplementary Figure I). Moreover, human clones derived from a single GFP-labeled cell started a synchronous beating and expressed cTnI after 48 hours of coculture with rat cardiomyocytes (Supplementary Movie human CSs 2a and 2a1 and Supplementary Figure II).
Furthermore, when BrdUrd was added to the culture medium, virtually all cells in the small CSs and those of the inner part of the largest CSs were labeled (Figure 3a), indicating that these cells were newly generated (Supplementary Figures III through Va).
Figure 3. CSs BrdUrd incorporation and CSs characterization. a, Fluorescence confocal analysis of BrdUrd-labeled human CSs for cardiac differentiation markers: 6-m scans (from the periphery to the center of the sphere) and final pictures (small and large images, respectively) of BrdUrd (green) and cTnI (red) (see Supplementary Figures III through V). b, Confocal analysis of human CSs after 12 hours of culture: CD-34, CD-31, KDR, and c-Kit labeling of CS-generating cells at the beginning of sphere formation. c, fluorescence-activated cell sorting analysis of postnatal mouse CSs-derived cells. A time course at 0 and 6 days was used, and the phenotype profile for CD34, cKit, Cd31, and sca-1 expression was analyzed and shown as a percentage of positive events. Data are presented as meanSD (n=3). *Statistically significant difference from 0 days. See the graphics in the Table and in Figure 6.
Human CS-generating cells were capable of self-renewal. With periodical dissociation, together with partial substitution of CGM every 2 to 3 days, a log-phase expansion of spheres was obtained (Figure 1b). Mouse CS growth was slower (probably because of the more differentiated features assumed in culture, such as beating) and serum-dependent as for the human CSs (Figure 1b).
As shown in Figure 3a and Supplementary Figure V, confocal immunofluorescence analysis of BrdUrd-labeled human CSs with anti-BrdUrd (green) and cardiac-troponin I (cTnI) or atrial natriuretic peptide (ANP) (red) revealed BrdUrd-positive cells, particularly in the inner of the spheres, whereas cTnI-positive or ANP-positive cells were mainly localized in the external layers. Similar features are shown in Supplementary Figures III and IV. BrdUrd-labeled cells (red) mostly localized in the center of a CS and colocalize with the Hoechst-labeled nuclei, whereas cardiac myosin heavy chain (MHC)-expressing cells (green) were preferentially located in the boundary layers. Furthermore, several CS cells expressed cardiac differentiation markers (cTnI, ANP) while still dividing, as indicated by BrdUrd incorporation (Figure 3a and Supplementary Figure Va), suggesting that early cardiac differentiation already occurred during the proliferation phase of their growth. Usually within 10 days, some spheres became adherent, showing a more flattened morphology. Some small cells eventually migrated out from these sun-like spheres in the form of adherent (differentiated) or small, round cells that could generate new spheres. After thawing from cryopreservation, CSs proliferated again, maintaining their ability to beat (Supplementary Movie: human CSs movie).
Phenotypic analysis of newly developing human and mouse CSs revealed expression of endothelial (KDR (human)/flk-1 [mouse], CD-31) and stem cell (CD-34, c-kit, sca-1) markers. As shown in Figure 3b, CSs at the 2- to 10-cell stage strongly reacted with antibodies against these antigens. In larger spheres, the expression pattern of some of these markers (particularly cKit) was similar to that of the BrdUrd-labeling (positive staining in the center and in some peripheral zones, generating satellite spheres; data not shown).
A time course (0 and 6 days) of the quantitative characterization of CS cells with these stem and endothelial markers was performed by fluorescence-activated cell sorting analysis (Figure 3c and Supplementary Figure VI). As shown at the beginning of their formation (0 days), the phenotype of these cells seems to reflect the epifluorescent microscopy analysis with 10% of positive staining for all four phenotypes. However, at 6 days, cKit appears to be the only conserved marker, suggesting that the cKit+ cells could be the main ones contributing to the maintenance of proliferation. The initial cell-labeling may reflect an early activation state, as has been suggested for CD-34 in several systems.17 Fluorescence microscopy analysis performed on cryosectioned human CSs revealed expression of cardiac differentiation markers (cTnI, MHC) and endothelial markers (von Willebrand factor) (Supplementary Figure Vc1 through Vc3). When totally or partially dissociated into single cells and cultured on collagen-coated dishes in the same medium as the explants, mouse and human CS-derived cells assumed a typical cardiomyocyte morphology, phenotype (Supplementary Figures Vb1 through Vb2 and VIIc and VIId), and function documented (in the mouse only) by spontaneous contraction (Supplementary Movie: mouse CSs movie 2a and 2b).
Human CSs did not beat spontaneously; however, these began to beat within 24 hours when cocultured with postnatal rat cardiomyocytes, losing their spherical shape and assuming a sun-like appearance. Markers of cardiac differentiation were coexpressed within GFP in labeled human CSs cells (Figure 2c).
To follow the differentiation process of CSs during the prenatal and postnatal age, MLC3F-nlacZ and cTnI-nlacZ mice were used.1112 These mice express a form of lacZ transgene that localizes within the nucleus under the skeletal and cardiac muscle myosin light chain or cardiac troponin I promoter, respectively. CSs obtained from embryonic day 9 to 12, fetal day 17 to 18, and from neonatal and adult mice showed spontaneous expression of the reporter gene in variable percentages (10% to 60%) of spheres in the different culture conditions used (Figure 4a1 through 4a4 and Supplementary Figure VIIa1, VIIa2, VIIb1, and VIIb2). Moreover, regarding the human ones, CS-generating cells from mice expressed stem (CD-34, sca-1, cKit) and endothelial cell markers (flk-1, CD-31) (data not shown).
Figure 4. CSs features in transgenic mice. a, Phase micrograph of CSs from MLC3F-nlacZ and cTnI-nlacZ mice. Nuclear lacZ expression is mainly localized in the external layers of embryo and adult CSs soon after their formation (inserts) and after a few days of culture (right and central panels) (see Supplementary Figure VII). b, Fluorescence analysis of a spontaneously differentiated mouse CS. As suggested from the synchronous contraction showen in culture (supplementary mouse CSs movie), cTnI (red) is expressed in the sphere and the migrated cells; in these, last sarcomers are also evident. c, Fluorescence and phase analysis of CSs from GFP-cKit, GFP-cKit/MLC3F-nLacZ, and GFP-cKt/cTnI-nlacZ mice. GFP-labeled cells were present a few minutes after their seeding in culture with CGM, at the beginning of the generation of the CSs, later in their inner mass, and after their migration out from the oldest adherent spheres (arrows) (upper left, lower left, and central panels). GFP-labeled cells did not colocalize with the blue-stained ones (arrows) in CSs from GFP-cKit/MLC3F-nLacZ and GFP-cKit/cTnI-nlacZ mice. Fluorescent cells also were present in the growth area of the CSs (arrows) (right upper and right lower panels). Fluorescence, phase (small), and merged (large) images.
On this basis, we used transgenic mice expressing GFP under the control of the c-kit promoter14 to further clarify the cellular origin of these spheres and to follow the pattern of their growth process. As shown in Figure 4c1, GFP-positive cells were present from the beginning of the formation of the CSs and, albeit with reduced fluorescence intensity, also later within the mass of cells of the CSs and in cells migrating from old adherent sun-like CSs (Figure 4c2). Moreover, as suggested by the growth pattern of human CSs, when satellite secondary CSs appeared to detach from the primary ones, GFP-positive cells localized on the margins of the latter and in the inner part of the former.
We studied this process in double-heterozygous mice obtained from GFP-cKit/MLC3F-nlacZ or GFP-cKit/cTnI-nLacZ crossings. As shown in Figure 4c3 and 4c4, -Gal positivity did not colocalize with GFP in cells present within the growing areas.
To investigate the survival and morpho-functional potential of the CSs in vivo, two sets of experiments were performed. In the first, CS cells were injected in the dorsal subcutaneous region of SCID mice. In the second, they were injected into the hearts of SCID beige mice, acutely after myocardial infarction. The objective of ectopic transplantation experiments was to study the pattern and the behavior of growth of CSs in a neutral milieu (ie, without specific cardiac induction) to verify their unique potential of generation of the main cardiac cell types and to exclude the potential of neoplastic transformation. For these experiments, 60 pooled spheres/inoculum/mouse from prenatal and postnatal MLC3F-nlacZ/B5-eGFP mice, TnI-nlacZ/B5-eGFP mice, MLC3F-nlacZ/CD-1 mice, and cTnI-nlacZ/CD-1 mice were used. During the first 10 days, beating was appreciable through the skin over the injection site, distant from large blood vessels. On day 17, animals were euthanized and the inoculum recognized as a translucent formation, grain-like in size, wrapped in ramified vessel-like structures. Observation of unfixed cryosections by fluorescence microscopy (Figure 5a1 through 5a4) revealed the presence of open spheres from which cells appeared to have migrated. Clusters of black holes, particularly in the periphery of the structure, were evident. The tissue contained tubular formations, surrounded by nuclei (Hoechst-positive), identified as cardiac sarcomeres by cTnI and sarcomeric myosin immunostaining (Figure 5b3 through 5b6). -Smooth muscle actin (-SMA)-positive structures (known to be transiently expressed during cardiomyogenesis)2,18 were present in the remainder of the spheres and associated with the vasculature (the clusters of black holes) (Figure 5a3 through 5a5). This exhibited well-differentiated structures with a thin endothelium expressing vascular endothelialcadherin (Figure 5b1) and a relative large lumen containing erythrocytes (Figure 5a3), indicating the establishment of successful perfusion by the host. Light microscopic observation of the inoculum, after X-gal staining, showed strong nuclear expression of striated muscle-specific lacZ in the remainder of the spheres and in some cells close to them (Figure 5b2). No multidifferentiated structures suggesting the presence of tumor formation were observed.
Figure 5. In vivo analysis (ectopic CSs inoculum). a1 to a5, Ectopic transplantation of CSs from MLC3F-nlacZ/B5-eGFP mouse to SCID mouse (upper left panels). Fluorescence analysis of unfixed cryosections (a1, a2, and a4) from the subcutaneous dorsal inoculum (day 17). GFP cells seemed to have migrated from the spheres, whereas clusters of vessel-like structures (a2) could be observed mainly in the external area. Staining for SMA of one of these cryosections showed positive immunoreaction of the sphere and some cells within the inoculum (a5). b-1 to b6, Fluorescence (b3 to b4) and phase analysis (b5 to b6) of fixed and immunostained cryosections from dorsal inoculum of CSs from MLC3F-nlacZ/CD-1 and cTnI-lacZ/CD-1 mice. Tubular structures were stained for sarcomeric myosin (b3 to b5) and cTnI (b4 through b6). X-Gal staining labeled the cells within and those migrating from CS (b2). Endothelial markers (SMA and vascular endothelialcadherin) stained the vasculature (black holes) (a3 and b1).
To test the acquisition of functional competence and the cardiac regenerative potential of the CSs when challenged into an infarcted myocardium, orthotopic transplantation experiments with human CSs were performed. To perform these, thawed (cryopreserved) adult human CSs from three atrial (one male and two female) and one ventricular (one female) biopsy specimens were injected into the viable myocardium bordering a freshly produced infarct. Each mouse received CSs from a single passage of an explant (derived from a single subject). Four control infarcted animals were injected with an equal volume of PBS. Eighteen days after the intervention, the animals were euthanized and infarct size was determined. Infarct size was 34.97.1 (SEM, 3.6) and 31.96.9 (SEM, 3.5) in the CS-treated group and PBS-injected group, respectively (P=NS). However, echocardiography showed better preservation of the infarcted anterior wall thickness in the CS-treated group compared with the PBS-injected group (0.800.29 [SEM, 0.15] versus 0.600.20 [SEM, 0.08]) (P=NS), particularly of percent fractional shortening (36.8516.43 [SEM, 8.21] versus 17.875.95 [SEM, 2.43]) (P
Figure 6. In vivo analysis (orthotopic transplantation of human CSs). Orthotopic transplantation performed in a SCID-beige mouse. Cryopreserved human CSs were transplanted into the viable myocardium bordering a freshly produced infarct. Confocal analysis of cryosectioned left ventricular heart 18 days after the coronary ligature shows that (a) cardiomyocytes expressing MHC (red) in the regenerating myocardium (particularly those indicated by the two central arrows) also stain positive for lamin A/C (green) (a specific human nuclear marker). In these cells, MHC expression is evident mainly in the perinuclear area (see Supplementary Figure X). Lamin A/C-labeled cells (red) are present in newly generated capillaries staining for -SMA (b1 through d), and platelet endothelial cell adhesion molecule (c). d, Confocal analysis of colocalization of lamin A/C-labeled cells (red) with the newly generated capillaries staining for -smooth muscle actin. e, Low-magnification image shows viable lamin A/C-expressing cells (green) in regenerating myocardium expressing MHC (red).
Myocardial Repair (Echocardiography)
At the time of evaluation, bands of regenerating myocardium were present (with different degrees of organization and thickness) throughout most of the infarcted areas, as evaluated with hematoxylineosin histochemistry (data not shown) and MHC immunofluorescence (Supplementary Figure IXa1 and IXa2). In the regenerating myocardium, cells expressing lamin A/C (a specific human nuclear marker) also colocalize with cardiomyocytes stained positive for MHC (Figure 6a and 6e and Supplementary Figures IXb1, IXb2, and X), newly generated capillaries stained for -SMA (Figure 6b1, 6b2, and 6d) and platelet endothelial cell adhesion molecule (Figure 6c), and with connexin-43expressing cells (data not shown).
CSs appear to be a mixture of cardiac stem cells, differentiating progenitors, and even spontaneously differentiated cardiomyocytes. Vascular cells were also present, depending on the size of the sphere and time in culture. It is possible that, as for neurospheres,19 differentiating/differentiated cells stop dividing and/or die, whereas stem cells continue to proliferate in an apparently asymmetric way, giving rise to many secondary spheres and to exponential growth in vitro. Mechanical dissociation favors this process. Death, differentiation, and responsiveness to growth factors of the different cells within the CSs could depend on the three-dimensional architecture and on localization within the CSs.20 The spontaneous formation of spheres is a known prerogative of neural stem cells, some tumor cell lines (LIM),21 endothelial cells,22 and fetal chicken cardiomyocytes.23 All these models (ours included) that mimic the true three-dimensional architecture of tissues consist of spheroids of aggregated cells that develop a two-compartment system composed of a surface layer of differentiated cells and a core of unorganized cells that first proliferate and then disappear over time (perhaps through apoptotic cell death). As well-documented in fetal chick cardiomyocytes and endothelial cell spheroid culture, three-dimensional structure affects the sensitivity of cells to survival and growth factors.21,22 In particular, central spheroid cells do not differentiate and are dependent on survival factors to prevent apoptosis, whereas the cells of the surface layer seem to differentiate beyond the degree that can be obtained in two-dimensional culture and become independent of the activity of survival factors.23 Furthermore, cellcell contact and membrane-associated factors, known to be important for the division of neural precursor cells,24 could be involved in our system. This is in accordance with the notion that stem cells (or cells with stem cell function) will only retain their pluripotency within an appropriate environment, as suggested by the niche hypothesis.25
Thus CSs can be considered clones of adult stem cells, maintaining their functional properties in vitro and in vivo after cryopreservation.
While the experiments performed for this article were ongoing, two articles were published concerning the isolation of cardiac stem cells or progenitor cells from adult mammalian hearts.26,27 Isolation of these cells was based exclusively on the expression of a stem cell-related surface antigen: c-kit in the first article and Sca-1 in the second one. In the first study,26 freshly isolated c-kit+ Lin cells from rat hearts were found to be self-renewing, clonogenic, and multi-potent, exhibiting biochemical differentiation into the myogenic cell, smooth muscle cell, or endothelial cell lineage but failing to contract spontaneously. When injected into an ischemic heart, these cells regenerated functional myocardium. In the second study,27 Sca-1+ cKit cells from mice hearts were induced in vitro to differentiate toward the cardiac myogenic lineage in response to 5-azacytidine. When given intravenously after ischemia/reperfusion, these cells targeted injured myocardium and differentiated into cardiomyocytes, with and without fusion with the host cells. Our data obtained on GFP-cKit transgenic mice also suggest that the adult cardiac stem cell is cKit+. It is possible that CSs enclose a mixed population of cells that, as in the niche, could promote the viability of cKit progenitors and contribute to their proliferation. The data obtained in the present article confirm the existence of adult cardiac stem cells/progenitor cells. More importantly, they demonstrate for the first time to our knowledge that it is possible to isolate cells from very small fragments of human myocardium and expand these cells in vitro many-fold (reaching numbers that would be appropriate for in vivo transplantation in patients) without losing their differentiation potential. Previously unforeseen opportunities for myocardial repair could now be identified.
Communicated by Eugene Braunwald, Harvard Medical School, Boston, MA, July 19, 2007 (received for review May 2, 2007)
The identification of cardiac progenitor cells in mammals raises the possibility that the human heart contains a population of stem cells capable of generating cardiomyocytes and coronary vessels. The characterization of human cardiac stem cells (hCSCs) would have important clinical implications for the management of the failing heart. We have established the conditions for the isolation and expansion of c-kit-positive hCSCs from small samples of myocardium. Additionally, we have tested whether these cells have the ability to form functionally competent human myocardium after infarction in immunocompromised animals. Here, we report the identification in vitro of a class of human c-kit-positive cardiac cells that possess the fundamental properties of stem cells: they are self-renewing, clonogenic, and multipotent. hCSCs differentiate predominantly into cardiomyocytes and, to a lesser extent, into smooth muscle cells and endothelial cells. When locally injected in the infarcted myocardium of immunodeficient mice and immunosuppressed rats, hCSCs generate a chimeric heart, which contains human myocardium composed of myocytes, coronary resistance arterioles, and capillaries. The human myocardium is structurally and functionally integrated with the rodent myocardium and contributes to the performance of the infarcted heart. Differentiated human cardiac cells possess only one set of human sex chromosomes excluding cell fusion. The lack of cell fusion was confirmed by the Cre-lox strategy. Thus, hCSCs can be isolated and expanded in vitro for subsequent autologous regeneration of dead myocardium in patients affected by heart failure of ischemic and nonischemic origin.
The recent identification of different classes of cardiac progenitor cells has suggested that the heart is not a terminally differentiated, postmitotic organ but an organ regulated by a stem cell compartment (1). The possibility has also been raised that stem cells are present in the normal and pathological human heart (2, 3). Together, these results point to a shift in paradigm concerning the biology of the heart and put forward potential therapeutic strategies for the failing heart. However, the actual existence of a human cardiac stem cell (hCSC) remains to be demonstrated. By definition, stem cells have to be self-renewing, clonogenic, and multipotent in vitro and in vivo (4, 5), and no studies to date have shown that the human heart contains primitive cells with these properties. Cells with limited growth and differentiation ability may acquire only the myocyte, endothelial cell (EC) or smooth muscle cell (SMC) lineage in vitro, and may not be capable of forming functionally competent myocardium in vivo. hCSCs have to be able to replace dead tissue with contracting myocardium composed of cardiomyocytes and vascular structures, independently from cell fusion. Heterokaryons divide poorly and have, at best, a transient positive impact on the age of the fused cells (6). Here, we report that these issues have been resolved, and hCSCs may represent a form of therapy for the diseased heart.
The documentation of hCSCs requires the identification of interstitial structures with the characteristics of stem cell niches and the recognition of the mechanisms of stem cell division that regulate niche homeostasis and the self-renewing properties of the human heart in vivo (7). We have found that the human heart contains clusters of hCSCs that are intimately connected by gap junctions and adherens junctions to myocytes and fibroblasts (Fig. 1 AC); myocytes and fibroblasts represent the supporting cells within the cardiac niches (7). Additionally, symmetric and asymmetric division of hCSCs was detected, respectively, by the uniform and nonuniform localization of the cell-fate determinants Numb and -adaptin (7) at one or both poles of hCSCs in mitosis (Fig. 1 D and E). The commitment to the myocyte lineage of hCSCs was also found within the niches. The coexpression of the stem cell antigen c-kit and myocyte transcription factors and sarcomeric proteins [see supporting information (SI) Fig. 6] is consistent with a lineage relationship between hCSCs and myocyte formation. C-kit POS cells expressing transcription factors for SMCs and ECs were also detected (data not shown). In the niches, hCSCs and committed cells were negative for hematopoietic markers and KDR (SI Table 1). These findings in the normal human heart, together with earlier observations in the diseased heart (3, 8), support the notion of a resident hCSC compartment that gives rise to the various cardiac cell progenies.
Cardiac niches and hCSC division. Sections of normal human myocardium. (AC) Cluster of c-kit POS cells (green). Arrows in A define the areas in B and C. Gap (connexin 43: Cx43, white; arrowheads) and adherens (N-cadherin: N-cadh, magenta; arrowheads) junctions are shown at higher magnification. Cx43 and N-cadh are present between c-kit POS cells and myocytes (-SA, red) and fibroblasts (procollagen, light blue); fibronectin, yellow. (D and E) Mitosis (phospho-H3, magenta; arrows) in c-kit POS cells; -adaptin (D, white) and Numb (E, yellow) show a uniform (D) and nonuniform (E) localization in the mitotic c-kit POS cells.
C-kit POS cells, i.e., hCSCs, were prepared with two methodologies. The first consisted of the enzymatic dissociation of myocardial samples from which c-kit POS cells were sorted with immunobeads and plated at low density (SI Fig. 7 AC ) to obtain multicellular clones from single founder cells. This procedure was dictated by the small size of the samples, 30 mg, which precluded FACS analysis at the outset. Successful isolation was obtained in 8 of 12 cases. The phenotype of the freshly isolated cells was characterized by FACS in 6 additional cases in which larger samples, 60 mg, were available. C-kit POS cells comprised 1.1 1.0% of the entire cell population. They were different from human bone marrow cells and were negative for markers of hematopoietic cells and KDR (Fig. 2 A and B; SI Table 2). Only small fractions of hCSCs expressed GATA4 and Nkx2.5, 0.5%.
Human CSCs. (A and B) Scatter plots of hCSCs (A) and human bone marrow cells (B). hCSCs do not express hematopoietic markers, KDR, GATA4, and Nkx2.5. (C) Nuclei (blue) of hCSCs were stained with a telomere probe (magenta). Lymphoma cells with short (7-kbp) and long (48-kbp) telomeres are shown for comparison. (D) Products of telomerase activity in hCSCs start at 50 bp and display a 6-bp periodicity. Samples treated with RNase and CHAPS buffer were used as negative controls, and HeLa cells were used as positive control. The band at 36 bp corresponds to an internal control for PCR efficiency. Optical density (arbitrary units, AU) is shown as mean SD.
With the second protocol, samples were cultured by the primary explant technique (SI Fig. 7 D and E ). Successful cell outgrowth was obtained in 46 of 70 cases. A monolayer of 6,000 cells was present at the periphery of each tissue aggregate, 3 weeks after seeding. C-kit POS cells accounted for 1.6 1.4%. Adherent cells at passage P0 were analyzed by immunocytochemistry and FACS (SI Fig. 8; SI Tables 1 and 2). In enzymatically dissociated cells, lineage negative (Lin) c-kit POS cells were 41 14%, and early committed cells (GATA4-positive) were 59 14%. Corresponding values with the primary explant technique were 52 12% and 48 12% (SI Fig. 9A ). In the presence of serum, hCSCs obtained with both protocols attached rapidly and continued to grow up to P8, undergoing 24 population doublings (PDs); the majority of experiments were concluded at P8. Cells maintained a stable phenotype and did not reach growth arrest. The percentage of c-kit POS cells did not vary from P1 to P8, averaging 71 8%. Undifferentiated cells constituted 63 6%. Ki67POS cycling-cells averaged 48 10%. p16INK4a, a cdk inhibitor, was present in 6 4% of the cells (SI Fig. 9 BD ). Thus, hCSCs are distinct from bone marrow cells and can be isolated and expanded in vitro.
To determine whether hCSCs reach senescence in culture, telomeric length was evaluated by Q-FISH (Fig. 2 C). From P3P4 (912 PDs) to P5P6 (1518 PDs) and P8P9 (2427 PDs), average telomere length in hCSCs decreased from 9.3 to 8.2 and 6.9 kbp, respectively (SI Fig. 10). From P3 to P9 there were 18 PDs with an average telomeric shortening of 130 bp per PD. This rate of telomere attrition is comparable with that of human bone marrow stem cells (9). Additionally, nearly 50% of the telomerase activity in hCSCs at P3P4 was still present at P8P9 (Fig. 2 D).
Critical telomere length associated with growth arrest and cellular senescence of hCSCs and human hematopoietic SCs varies from 2.0 to 1.5 kbp (3, 9). The fraction of hCSCs with critical telomeric shortening increased from 1% at P3P4 to 2% at P5P6 and 5% at P8P9. However, after 2427 PDs at P8P9, 69% hCSCs had telomere length 5.0 kbp (SI Fig. 10). It can be predicted that cells at P8P9 can undergo 23 additional PDs (52 = 3kbp/0.13kbp = 23 PDs) before irreversible growth arrest (10). In theory, 50 PDs can result in the formation of 1 1015 hCSCs before replicative senescence is reached. Thus, hCSCs can be extensively grown in vitro in the absence of a major loss in their expansion potential.
hCSCs obtained by enzymatic digestion and explant technique were plated at limiting dilution and in Terasaki plates, respectively. In the first case, 1,530 c-kit POS cells were seeded, and after 34 weeks, 11 clones were generated. In the second case, cells were placed in individual wells, and 53 clones were formed from 6,700 seeded cells. Thus, hCSCs had 0.70.8% cloning efficiency (Fig. 3 AC). Clones were expanded and characterized. Doubling time was 29 10 h, and 90 7% of cells after 5 days were BrdUPOS. Clonogenic hCSCs retained largely their primitive state and were negative for hematopoietic markers, KDR, and transcription factors and cytoplasmic proteins of cardiac cells (SI Fig. 11 A and B; SI Table 1).
In vitro properties of hCSCs. (AC) Clones formed by hCSCs (c-kit, green) isolated by enzymatic digestion (A and C) or primary explant (B). The number of cells increased with time (C). (D) hCSCs generate myocytes positive for cardiac myosin heavy-chain (MHC), -SA, and -cardiac-actinin (-actinin). Sarcomeres are apparent (Insets); phalloidin, green. (E) Myocyte shortening in cells derived from clonogenic hCSCs was recorded by two-photon microscopy and laser line-scan imaging (Left). The line scan is shown (Right), and arrowheads point to individual contractions. (F) Myocytes derived from EGFP-positive hCSCs, cocultured with neonatal myocytes. EGFP-positive human myocytes shorten (arrowheads) with electrical stimulation. (G) Calcium transients in EGFP-positive human myocytes and EGFP-negative rat myocytes (calcium indicator Rhod-2, red).
In differentiating medium, hCSCs gave rise to myocytes, SMCs, and ECs (Fig. 3 D; SI Fig. 11 C and D ). Developing myocytes had sarcomere striation (Fig. 3 D) and, after electrical stimulation at 1 Hz, showed contractile activity (Fig. 3 E). Moreover, hCSCs were infected with a lentivirus expressing EGFP and cocultured with neonatal rat myocytes. Two weeks later, cultures were stimulated, and 9% shortening of EGFP-positive human myocytes was detected (Fig. 3 F). In the presence of the calcium indicator Rhod-2, calcium transient was identified in EGFP-positive human myocytes and EGFP-negative rat myocytes (Fig. 3 G). Thus, hCSCs form multicellular clones and differentiate into contracting myocytes.
Nonclonogenic hCSCs, collected from eight patients, were injected in the infarcted mouse or rat heart to form chimeric organs containing human myocytes and coronary vessels. Cell treatment led to areas of myocardial regeneration that were located within the infarct and were positive for -sarcomeric actin (-SA) and human AluDNA sequences (Fig. 4 A). Human myocardium was found in 17 of 25 treated mice (68%), and 14 of 19 treated rats (74%). hCSCs were delivered with rhodamine-labeled microspheres for the recognition of the sites of injection and correct administration of cells (1). The absence of myocardial regeneration was due to technical failure to properly inject hCSCs in the rodent heart. Conversely, successful cell implantation was invariably associated with the presence of human myocardium.
hCSCs regenerate infarcted myocardium. (A) Mouse heart 21 days after infarction and injection of hCSCs. Human myocardium (arrowheads) is present within the infarct (MI). BZ, border zone. Areas in rectangles are shown at higher magnification below. Human myocytes are -SA- (red) and Alu- (green) positive. Asterisks indicate spared myocytes. (B) Expression of human (h) genes by real-time RT-PCR in treated infarcted rats at 511 and 1221 days. Clonogenic hCSCs were used for comparison of human transcripts. (C) Electrophoresis of real-time RT-PCR products (for sequences see SI Fig. 11J ).
The human myocardium comprised 1.3 0.9 mm3 in mice and 3.7 2.9 mm3 in rats. Accumulation of new cells was also determined by BrdU labeling because BrdU was given to the animals throughout the experiment (SI Fig. 12 A and B ). The human myocardium consisted of myocytes that occupied 84% of the tissue, whereas arterioles and capillaries accounted for 8%. The human origin of the myocardium was confirmed by the detection of human Alu and Mlc2v DNA by PCR in sections of regenerated infarcts (SI Fig. 12C ). PCR products had the expected molecular weight, and the nucleotide sequences confirmed the specificity of the assay (SI Fig. 12 DF ).
Three control groups were used: (i) unsuccessfully treated-animals (eight mice, five rats); (ii) immunodeficient infarcted mice (n = 12) and immunosuppressed infarcted rats (n = 9) injected with PBS; and (iii) immunosuppressed infarcted rats injected with c-kit-negative cells obtained from the unfractionated cell population at P1 (n = 16). Infarct size was similar in all groups: 48 9% in mice and 54 11% in rats. Myocardial regeneration was absent in control hearts with the exception of 3 of the 16 hearts treated with c-kit-negative cells. In one case, a few -SA and Alu-positive cells were found within the infarct, whereas, in the other two, a small band of human myocardium was identified near the border zone (SI Fig. 12 G and H ).
For completeness, clonogenic hCSCs were injected in infarcted rats shortly after coronary ligation to determine their multipotentiality in vivo (n = 6) and establish whether multipotentiality persisted when cell implantation was performed 5 days after coronary occlusion under the condition of a fully developed ischemic injury (n = 10). In both cases, clonogenic hCSCs regenerated the infarcted myocardium (SI Fig. 12I ) by forming human myocytes and coronary vessels (see below).
Real-time RT-PCR was used to demonstrate human transcripts for myocyte (MLC2v, connexin 43), SMC (smooth-muscle myosin heavy-chain 11 = Mhc 11) and EC (vWF) genes, and the housekeeping gene GAPDH in infarcted rat hearts treated with clonogenic hCSCs. Because there is no baseline in the rat myocardium for the analysis of human genes, clonogenic hCSCs were used for comparison (n = 4). With respect to clonogenic hCSCs, there was a substantial up-regulation of human myocardial transcripts for parenchymal and vascular cells in the infarcted heart (Fig. 4 B). The expression of human MLC2v, connexin 43, Mhc 11, and vWF increased from 511 days (n = 8) to 1221 days (n = 15) after infarction and cell implantation. RT-PCR products had the expected molecular weight (Fig. 4 C), and the nucleotide sequences confirmed the specificity of the assay (SI Fig. 12J ). Thus, hCSCs generate human myocardium.
After the identification of AluDNA, cardiac myosin heavy-chain (MHC), troponin I, and -SA were detected in human myocytes. Moreover, GATA4, MEF2C, connexin 43, and N-cadherin were identified (SI Figs. 13 and 14). Human myocytes varied in size from 100 to 2,900 m3 (SI Fig. 14). Human coronary arterioles and capillaries were also found (SI Figs. 13 and 14). The number of human arterioles and capillaries was comparable in rats and mice; there was one capillary/eight myocytes (SI Fig. 14), and the diffusion distance for oxygen averaged 18 m. These parameters are similar to those found in the late fetalneonatal human heart. Thus, hCSCs differentiate into human myocytes and coronary vessels, leading to the formation of a chimeric heart.
Two protocols were used to test whether the generation of human myocardium involved fusion events between hCSCs and rodent cells. hCSCs were infected with a lentivirus carrying Cre-recombinase (infection efficiency = 90%) and injected in the infarcted heart of mice expressing loxP-flanked EGFP (n = 6). If fusion were to occur, EGFP transcription would be activated in the recipient cells by Cre-mediated excision of the stop codon in the EGFP promoter (1). At 10 days after infarction and cell implantation, newly formed human myocardial cells showed a nuclear localization of Cre protein (Fig. 5 A; SI Fig. 15). However, human myocytes and vessels were negative for EGFP, indicating that the formation of heterokaryons was not involved in cardiac repair.
Integration of human myocardium. (A) Human myocytes are Cre-recombinase-positive (white) but EGFP negative. (B) Human myocytes and vessels show, at most, two human X-chromosomes (X-Chr, white dots; arrowheads). Mouse X-Chr (magenta dots; arrows) are present in myocytes of the border zone (BZ). (C) Transmural infarct in a treated rat; human myocardium (arrowheads) is present within the infarct. The area in the rectangle is shown at higher magnification (Bottom); human myocytes are -SA- (red) and Alu- (green) positive. Echocardiogram shows contraction in the infarcted wall (arrowheads). (D) Ventricular function. Results are mean SD. * and , Difference, P
The second protocol consisted of the evaluation of the number of sex chromosomes by Q-FISH in human myocytes and coronary vessels. Because female human cells were injected in female mice and rats, human, rat and mouse X-chromosomes were measured. We never found a colocalization of a human X-chromosome with a mouse or rat X-chromosome in regenerated myocytes and vessels (Fig. 5 B). Human myocytes, SMCs, and ECs carried, at most, two human X-chromosomes. Thus, hCSCs form human myocardium independently from cell fusion.
Echocardiograms were examined retrospectively after the histological documentation of transmural infarcts and the presence of human myocardium (Fig. 5 C; SI Fig. 16 AC ). Tissue regeneration restored partly contractile function in the infarct, resulting in an increase of ejection fraction (SI Fig. 16D ), attenuation of chamber dilation (SI Fig. 16E ), and improvement of ventricular function (Fig. 5 D).
The interaction between human and rodent myocardium was determined by an ex vivo preparation and two-photon microscopy. EGFP-positive hCSCs were injected in infarcted mice, and the heart was studied 2 weeks later (n = 6). After the blockade of contraction and spontaneous activity, the heart was perfused with Rhod-2 and stimulated at 1 Hz. Calcium transient was recorded in EGFP-positive human myocytes and EGFP-negative mouse myocytes. The synchronicity in calcium tracings between these myocyte populations documented their functional integration (Fig. 5 E). hCSC-derived myocytes acquired the properties of the recipient rodent myocardium, indicating that primitive cells of human origin possess a high level of plasticity. Additionally, connexin 43 was found between human and rodent myocytes (Fig. 5 F) demonstrating their structural coupling. Thus, both myocardial components of the chimeric heart participate in the performance of the infarcted heart.
The current work demonstrates that the human heart possesses a pool of clonogenic hCSCs that can acquire the myocyte, SMC, and EC lineages in vitro and in vivo. The ability of hCSCs to create cardiomyocytes and coronary vessels in vivo provides strong evidence in favor of the role that hCSCs have in cardiac homeostasis and myocardial regeneration. Besides their therapeutic implications, these observations challenge the view of the heart as a postmitotic organ (11) and form the basis of a paradigm in which multipotent hCSCs modulate the physiological turnover of the heart. Understanding the mechanisms of cardiac homeostasis would offer the opportunity to potentiate this process and promote cardiac repair after injury.
Human cells with the ability to differentiate into cardiomyocytes have been obtained from myocardial biopsies and were claimed to possess the properties of stem cells (2). These cells express the typical markers of human circulating endothelial-progenitor cells (EPCs): CD34, CD31, and KDR, together with c-kit (12). The expression of CD34, CD31, and KDR does not compromise the ability of these circulating cells to acquire the myocyte lineage in vitro (13) and in vivo (14). The presence of these epitopes, however, suggests that these cells originate from the bone marrow and only subsequently accumulate within the heart. These early findings failed to provide evidence for the clonogenicity of these cells in vitro and their multilineage differentiation in vivo, which are critical for the recognition of a tissue-specific adult stem cell (1). The inability of these cells to generate a functional human myocardium in vivo is consistent with the role of EPCs in cardiac repair; they acquire, at low efficiency, the myocyte lineage and exert a paracrine effect on the infarcted heart (13).
Conversely, as demonstrated here, hCSCs are positive for the stem cell antigen c-kit but are negative for the hematopoietic and endothelial antigens CD45, CD34, CD31, and KDR; CD45 and KDR are typically expressed in a subset of bone marrow c-kit POS cells that have the ability to migrate to the heart after injury (12). Stem cell niches have been identified here in the normal human myocardium, and hCSCs divide symmetrically and asymmetrically and give rise to differentiating and lineage-negative cells. This provides evidence in favor of a linear relationship between hCSCs and myocyte formation. Additionally, these observations do not support the notion of dedifferentiation of mature myocytes with the acquisition of a stem cell phenotype. Importantly, clonogenic hCSCs have the inherent potential to form contracting myocardium integrated structurally and functionally with the recipient heart. Although CSCs with similar characteristics were shown in animal models (4, 5), the applicability of this information to humans was seriously questioned and considered a major limitation for the clinical implementation of CSCs (15).
In the current study, three possibilities were considered in the formation of human myocardium within the infarcted mouse and rat heart (1): (i ) Growth and differentiation of hCSCs; (ii ) Fusion of hCSCs with the surviving mouse or rat cardiac cells, followed by proliferation of the heterokaryons and generation of myocytes and coronary vessels; and (iii ) A combination of these two processes. The evaluation of human, mouse, and rat sex chromosomes together with the Cre-lox strategy has indicated that the generation of human myocardium involved only the commitment of hCSCs to cardiomyocytes, SMCs, and ECs. The unlikely involvement of cell fusion was supported by the size (1002,900 m3) of human myocytes. If fusion were to be implicated, the newly formed human myocytes should have had a volume of at least 25,000 m3 or larger, that is, the volume of adult mouse and rat cardiomyocytes. It is improbable that fusion of a primitive cell with a terminally differentiated myocyte can induce division of a highly specialized and rapidly contracting cell permanently withdrawn from the cell cycle (1, 6).
The identification of a resident hCSC pool in the human heart is apparently at variance with the small foci of myocardial regeneration present after acute and chronic infarcts or pressure overload in patients (3, 16). The limitation that resident hCSCs have in reconstituting myocardium after infarction has been interpreted as the unequivocal documentation of the inability of the adult heart to create cardiomyocytes (11). The inevitable evolution of ischemic injury is myocardial scarring with loss of mass and contractile function. A possible explanation of this apparent paradox has been obtained in animal models of the human disease (5). Stem cells are present throughout the infarcted myocardium but, despite the postulated resistance of these cells to death stimuli, they follow the same pathway of cardiomyocytes and die by apoptosis. The fate of hCSCs is comparable with that of the other cells, and myocyte formation is restricted to the viable portion of the human heart (3).
It might come as a surprise, but a similar phenomenon occurs in solid and nonsolid organs, including the skin, liver, intestine, and kidney. In all cases, occlusion of a supplying artery leads to scar formation mimicking cardiac pathology (1720). In the presence of polyarteritis nodosa and vasculitis, microinfarcts develop in the intestine and skin, and resident SCs do not repair the damaged organs (21). In nonsolid organs, infarcts of the bone marrow are seen with sickle cell anemia (21). Thus, the SC compartment appears to be properly equipped to modulate growth during postnatal development and regulate homeostasis in adulthood. However, SCs do not respond effectively to ischemic injury or, late in life, to aging and senescence of the organ and organism (22, 23).
Current knowledge supports the notion that primitive cells are present in the heart at the very beginning of embryonic life and regulate heart morphogenesis and postnatal development (24). By introducing the EGFP gene in the mouse embryo, at the stage of the morulablastocyst transition, the patterns of myocardial histogenesis have been defined and the presence of a common progenitor of cardiomyocytes in prenatal and postnatal life suggested (24). The documentation of myocardial specification of embryonic stem cells (25, 26), in particular c-kit POS Nkx2.5POS cells (26), supports the view that a pool of resident c-kit POS progenitors is implicated in cardiac morphogenesis. These findings are consistent with the existence of a pool of primitive cells in the adult human heart.
hCSCs have been isolated expanded and characterized in vitro and in vivo after implantation in the infarcted rodent heart. Protocols are described in SI Materials and Methods .
The lentivirus expressing Cre-recombinase was kindly provided by Drs. Chang and Terada (University of Florida, Gainesville, FL) and the lymphoma cells by Dr. Blasco (Spanish National Cancer Centre, Madrid, Spain). This work was supported by National Institutes of Health grants.
Author contributions: C.B., M.R., T.H., R.W.S., K.U., R.B., J.K., A.L., and P.A. designed research; C.B., M.R., T.H., J.T., A.N., A.D.A., S.Y.-A., I.T., R.W.S., N.L., S.C., A.P.B., D.A.D., E.Z., F.Q., K.U., R.E.M., J.K., and A.L. performed research; C.B., M.R., T.H., J.T., A.N., A.D.A., S.Y.-A., I.T., R.W.S., N.L., D.A.D., K.U., R.E.M., R.B., J.K., A.L., and P.A. analyzed data; and C.B., M.R., J.K., A.L., and P.A. wrote the paper.
Conflict of interest statement: P.A. has applied for a patent.
This article contains supporting information online at http://www.pnas.org/cgi/content/full/0706760104/DC1.
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Human cardiac stem cells – PNAS
Researchers at the University of California, Berkeley, in collaboration with scientists at the Gladstone Institutes, have developed a template for growing beating cardiac tissue from stem cells, creating a system that could serve as a model for early heart development and a drug-screening tool to make pregnancies safer.
In experiments to be published Tuesday, July 14, in the journal Nature Communications, the researchers used biochemical and biophysical cues to prompt stem cells to differentiate and self-organize into micron-scale cardiac tissue, including microchambers.
“We believe it is the first example illustrating the process of a developing human heart chamber in vitro,” said Kevin Healy, a UC Berkeley professor of bioengineering, who is co-senior author of the study with Dr. Bruce Conklin, a senior investigator at the Gladstone Institute of Cardiovascular Disease and a professor of medical genetics and cellular and molecular pharmacology at UC San Francisco. “This technology could help us quickly screen for drugs likely to generate cardiac birth defects, and guide decisions about which drugs are dangerous during pregnancy.”
Screening for drug toxicity
To test the potential of the system as a drug-screening tool, the researchers exposed the differentiating cells to thalidomide, a drug known to cause severe birth defects. They found that at normal therapeutic doses, the drug led to abnormal development of microchambers, including decreased size, problems with muscle contraction and lower beat rates compared with heart tissue that had not been exposed to thalidomide.
“We chose drug cardiac developmental toxicity screening to demonstrate a clinically relevant application of the cardiac microchambers,” said Conklin. “Each year, as many as 280,000 pregnant women are exposed to drugs with evidence of potential fetal risk. The most commonly reported birth defects involve the heart, and the potential for generating cardiac defects is of utmost concern in determining drug safety during pregnancy.”
The new milestone comes nearly four months after Healy and other UC Berkeley researchers publicly debuted a system of beating human heart cells on a chip that could be used to screen for drug toxicity. However, that heart-on-a-chip device used pre-differentiated cardiac cells to mimic adult-like tissue structure.
In this new study, the scientists mimicked human tissue formation by starting with stem cells genetically reprogrammed from adult skin tissue to form small chambers with beating human heart cells. Conklin’s lab at Gladstone, an independent, nonprofit life science research organization affiliated with UC San Francisco, supplied these human induced pluripotent stem cells for this study.
The undifferentiated stem cells were then placed onto a circular-patterned surface that served to physically regulate cell differentiation and growth.
Location, location, location
By the end of two weeks, the cells that began on a two-dimensional surface environment started taking on a 3D structure as a pulsating microchamber. Moreover, the cells had self-organized based upon whether they were positioned along the perimeter or in the middle of the colony.
Compared with cells in the center, cells along the edge experienced greater mechanical stress and tension, and appeared more like fibroblasts, which form the collagen of connective tissue. The center cells, in contrast, developed into cardiac muscle cells. Such spatial organization was observed as soon as the differentiation started. Center cells lost the expression of octamer-binding transcription factor 4 (OCT4) and epithelial cadherin (E-cadherin) faster than perimeter cells, which are critical to the development of heart tissue.
“This spatial differentiation happens in biology naturally, but we demonstrated this process in vitro,” said study lead author Zhen Ma, a UC Berkeley postdoctoral researcher in bioengineering. “The confined geometric pattern provided biochemical and biophysical cues that directed cardiac differentiation and the formation of a beating microchamber.”
Could eventually replace animal models
Modeling early heart development is difficult to achieve in a petri dish and tissue culture plates, the study authors said. This area of study has typically involved the dissection of animals at different stages of development to study the formation of organs, and how that process can go wrong.
“The fact that we used patient-derived human pluripotent stem cells in our work represents a sea change in the field,” said Healy. “Previous studies of cardiac microtissues primarily used harvested rat cardiomyocytes, which is an imperfect model for human disease.”
The researchers pointed out that while this study focused on heart tissue, there is great potential for use of this technology to study other organ development.
“Our focus here has been on early heart development, but the basic principles of patterning of human pluripotent stem cells, and subsequently differentiating them, can be readily expanded into a broad range of tissues for understanding embryogenesis and tissue morphogenesis,” said Healy.
The National Institutes of Health and a Siebel Postdoctoral Fellowship helped support this research.
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Growing beating cardiac tissue from stem cells: New model …
We present a strategy to optimize cardiac differentiation in suspension for hiPSCs.
The matrix-free suspension platform integrates hPSC expansion and differentiation.
Cardiac production in suspension achieves >90% purity with 1L spinner flasks.
The production process in suspension is defined, scalable, and GMP compliant.
To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM) for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC) aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2109 CM/L at scales up to 1L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.
Myocardial infarction and heart failure are leading causes of death worldwide. As the myocardium has a very limited regenerative capacity, endogenous cell regeneration cannot adequately compensate for heart damage caused by myocardial infarction. The concept of cell replacement therapy is an appealing approach to the treatment of these cardiac diseases. HPSCs are an attractive cell source for cell replacement therapies because they can be expanded indefinitely in culture and efficiently differentiated into a variety of cell lineages, including cardiac cells. However, current hPSC expansion and differentiation methods rely on adherent cell culture systems that are challenging to scale up to the levels required to support pre-clinical and clinical studies.
Activin/Nodal/TGF-, BMP, and Wnt signaling play pivotal roles in regulating mesoderm and cardiac specification during embryo development (Arnold and Robertson, 2009, Buckingham et al., 2005, Tam and Loebel, 2007, David et al., 2008, Naito et al., 2006, Ueno et al., 2007andBurridge et al., 2012). Significant progress has been made in the cardiac differentiation process by modulating Activin, BMP, and Wnt pathways, which can efficiently drive differentiation to over 80% purity of CM (Burridge et al., 2014, Kattman et al., 2011, Lian et al., 2012, Yang et al., 2008, Zhang et al., 2012andZhu et al., 2011). Using an adherent cell culture platform, one study revealed that using 2 small Wnt pathway modulators to sequentially activate and then inhibit Wnt signaling at different differentiation stages of the culture is sufficient to drive cardiac differentiation and generate CM with high purity (Lian et al., 2012). In spite of this, adherent culture systems have limited scalability and are not practical to support the anticipated CM requirements of clinical trials. Alternatively, using an embryoid body (EB) differentiation method, a complex cardiac induction procedure involving stage-specific treatments with growth factors and small molecules to modulate Activin/Nodal, BMP, and Wnt pathways has been reported to be effective in cardiac differentiation in a suspension culture system (Kattman et al., 2011andYang et al., 2008). However, the process of generating EBs is inefficient, rendering this method impractical for large scale CM production. An additional limitation of these approaches for scale-up application is that both methods are based on the expansion of the hPSCs in adherent culture and the subsequent CM differentiation process in either adherent culture or as EBs. The labor intensiveness and limited scalability of current processes have been the primary bottle necks to the large scale production of CM for clinical applications of hPSC-derived CM.
Pre-clinical studies suggest that doses of up to one billion CM will be required to achieve therapeutic benefit after transplantation (Chong et al., 2014andLaflamme and Murry, 2005). In order to meet the current CM demand for pre-clinical studies and the anticipated demand for foreseeable clinical studies, development of a robust, scalable and cGMP-compliant differentiation process for the production of both hPSCs and hPSC-derived CM is essential. Suspension cell culture is an attractive platform for large scale manufacture of cell products for its scale-up capacity. Application of a suspension culture platform to support hPSC growth in matrix-free cell aggregates has been well established (Amit et al., 2010, Krawetz et al., 2010, Olmer et al., 2010, Singh et al., 2010, Steiner et al., 2010andChen et al., 2012). We previously also reported the development of a defined, scalable and cGMP-compliant suspension system to culture hPSCs in the form of cell aggregates (Chen et al., 2012). With this suspension culture system, hPSC cultures can be serially passaged and consistently expanded. In the present study we adapted our suspension culture system to establish a robust, scalable and cGMP-compliant process for manufacturing CM. We were able to use hPSC aggregates generated in the suspension culture system directly to produce CM with high efficiency and yield in suspension with various scales of spinner flasks. We optimized various critical process parameters including: small molecule concentration, induction timing and agitation rates for differentiation cultures in spinner flasks with scales up to 1L. In this study, we integrated undifferentiated hPSC expansion and small molecule-induced cardiac differentiation into a scalable suspension culture system using spinner flasks, providing a streamlined and cGMP-compliant process for scale-up CM differentiation and production.
We routinely maintained the hPSCs lines H7 (WA07, WiCell), ESI-017 (BioTime), and a hiPSC line (a gift from Dr. Joseph Wu, Stanford) in the form of cell aggregates in suspension culture as previously described (Chen et al., 2012). Briefly, suspension-adapted hPSCs were seeded as single cells at a density of 2.53105 cells/mL in 125, 500, or 1000mL spinner flasks (Corning) containing culture medium (StemPro hESC SFM, Thermo Fisher Scientific, Life Technologies) with 40ng/mL bFGF (Life Technologies) and 10M Y27632 (EMD Millipore). Stirring rates were adjusted to between 5070rpm depending on the vessel size and hPSC line. Medium was changed every day by demi-depletion with fresh culture medium without Y27632. Cells were dissociated with Accutase (Millipore) into single cells and passaged every 34days when the aggregate size reached approximately 300m. Cell suspension cultures were maintained in 5% CO2 with 95% relative humidity at 37C.
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Development of a scalable suspension culture for cardiac …
The notion of the adult heart as terminally differentiated organ without self-renewal potential has been undermined by the existence of a subpopulation of replicating myocytes in normal and pathological states. The origin and significance of these cells has remained obscure for lack of a proper biological context. We report the existence of Lin c-kitPOS cells with the properties of cardiac stem cells. They are self-renewing, clonogenic, and multipotent, giving rise to myocytes, smooth muscle, and endothelial cells. When injected into an ischemic heart, these cells or their clonal progeny reconstitute well-differentiated myocardium, formed by blood-carrying new vessels and myocytes with the characteristics of young cells, encompassing 70% of the ventricle. Thus, the adult heart, like the brain, is mainly composed of terminally differentiated cells, but is not a terminally differentiated organ because it contains stem cells supporting its regeneration. The existence of these cells opens new opportunities for myocardial repair.
Until recently, the accepted paradigm in cardiac biology considered the adult mammalian heart to be a postmitotic organ without regenerative capacity. It has been assumed that from shortly after birth to adulthood and senescence the heart has a relatively stable but slowly diminishing number of myocytes. This static view of the myocardium implied that both myocyte death and myocyte regeneration had little role in cardiac cellular homeostasis. Although stem cells have been isolated from many adult tissues including the blood, skin, central nervous system, liver, gastrointestinal tract, and skeletal muscle (see Rosenthal, 2003), the search for a cardiac stem cell has been considered futile given the accepted lack of regenerative potential of this tissue.
Evidence challenging the accepted wisdom has been slowly accumulating McDonnell and Oberpriller 1984andRumyantsev and Broisov 1987. In the past few years, we have documented the existence of cycling ventricular myocytes in the normal and pathologic adult mammalian heart of several species, including humans Kajstura et al. 1998, Beltrami et al. 2001andQuaini et al. 2002. Although these data provided an alternative view of cardiac homeostasis, they also raised questions because it required reconciliation of two apparent contradictory bodies of evidence: the well-documented irreversible withdrawal of cardiac myocytes from the cell cycle soon after birth on one hand MacLellan and Schneider 2000andChien and Olson 2002, and the presence of cycling myocytes undergoing mitosis and cytokinesis on the other. These results raised the question as to the origin of the cycling myocytes and their dramatic increase in response to an acute work overload.
In cases of sex-mismatched cardiac transplants in humans, the female hearts in the male hosts had a significant number of Y positive myocytes and coronary vessels (Quaini et al., 2002). Most likely due to technical differences (Anversa and Nadal-Ginard, 2002a), there are some discrepancies among groups about the degree of cardiac chimerism Muller et al. 2002, Glaser et al. 2002andLaflamme et al. 2002. It is likely that these male cells colonized the female heart after the transplant and subsequently differentiated, although alternative explanations have been raised. These male cells in the female heart presuppose the existence of mobile stem-like cells able to differentiate into the three main cardiac cell types: myocytes, smooth, and endothelial vascular cells.
Primitive cells of donor and recipient origin that express stem cell-related surface antigensc-kit, Sca-1, and MDR1were identified in the recipient hearts. More importantly, identical cells were found in human control hearts Quaini et al. 2002andAnversa and Nadal-Ginard 2002b. It is well known that in early fetal life, c-kitPOS cells colonize the yolk sack, liver, and probably other organs. The colonized organs express stem cell factor (SCF), the ligand of the c-kit receptor (Teyssier-Le Discorde et al., 1999); SCF mRNA is also present in fetal and neonatal myocardium (Kunisada et al., 1998), raising the possibility that stem-like cells could have been in the heart from fetal life. The rapid induction of SCF during myocardial ischemia (Frangogiannis et al., 1998) could be involved in the activation of these cells and explain the significant increase in new myocyte formation (Beltrami et al., 2001). However, the origin of these primitive cells, their presence in normal and pathological hearts, together with the identification of some of them having initiated the cardiomyocyte gene expression program, is suggestive that they might be true cardiac stem cells that give rise to the cycling myocytes detected in the adult heart. If this were the case, their manipulation might provide the opportunity to stimulate myocardial regeneration with endogenous cells. For this reason, we endeavored to establish a precursor-product relationship between these primitive cells and the fully differentiated cardiac cells and to determine, in vitro and in vivo, whether they behave like true adult cardiac stem cells.
To determine whether the putative cardiac stem cells detected in human heart transplants and their controls are bona fide stem cells with cardiogenic potential, we isolated them to test their differentiation potential in vivo and in vitro. For experimental convenience, we chose the rat as the animal model system. We first analyzed whether cells with the cell surface markers commonly expressed by other stem cells could be identified in the adult rat myocardium. Based on the postulated higher number of proliferating stem and precursor cells with age (Morrison et al., 1996), we analyzed the myocardium from older animals. Histological sections of myocardium from Fisher rats 2023 months of age were examined by confocal microscopy for the presence of cells negative for the expression of blood lineage markers (Lin) but positive for the common stem cell markers c-kit (Kondo et al., 2003), Sca-1 (Morrison et al., 1997), and MDR-1 (Sellers et al., 2001). Small Lin cells with a very high nucleus/cytoplasm ratio and positive for each of the above markers were distributed throughout the ventricular and atrial myocardium with a higher density in the atria and the ventricular apex. Because of the role of bone marrow-derived Lin c-kitPOS cells in myocardial regeneration (Orlic et al., 2001), the mesodermal origin of both the heart and the bone marrow, and the use of c-kit as a hematopoietic stem cell marker Morrison et al. 1997, Weissman et al. 2001andKondo et al. 2003, we decided to concentrate on the cardiac cells expressing this marker, the receptor for SCF. Although the density of these cells varied among different regions of the heart, on average we identified one Lin c-kitPOS cell every 1 104 myocytes. It should be noted that most, if not all, of the detected c-kitPOS cells were negative for the pan leukocyte marker CD45 and the endothelial/hematopoietic progenitor marker CD34.
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Adult Cardiac Stem Cells Are Multipotent and Support …
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Stem Cells News — ScienceDaily
Cardiac muscle cells or cardiomyocytes (also known as myocardiocytes or cardiac myocytes) are the muscle cells (myocytes) that make up the cardiac muscle. Each myocardial cell contains myofibrils, which are specialized organelles consisting of long chains of sarcomeres, the fundamental contractile units of muscle cells. Cardiomyocytes show striations similar to those on skeletal muscle cells. Unlike multinucleated skeletal cells, the majority of cardiomyocytes contain only one nucleus, although they may have as many as four. Cardiomyocytes have a high mitochondrial density, which allows them to produce adenosine triphosphate (ATP) quickly, making them highly resistant to fatigue.
There are two types of cells within the heart: the cardiomyocytes and the cardiac pacemaker cells. Cardiomyocytes make up the atria (the chambers in which blood enters the heart) and the ventricles (the chambers where blood is collected and pumped out of the heart). These cells must be able to shorten and lengthen their fibers and the fibers must be flexible enough to stretch. These functions are critical to the proper form during the beating of the heart.
Cardiac pacemaker cells carry the impulses that are responsible for the beating of the heart. They are distributed throughout the heart and are responsible for several functions. First, they are responsible for being able to spontaneously generate and send out electrical impulses. They also must be able to receive and respond to electrical impulses from the brain. Lastly, they must be able to transfer electrical impulses from cell to cell.
All of these cells are connected by cellular bridges. Porous junctions called intercalated discs form junctions between the cells. They permit sodium, potassium and calcium to easily diffuse from cell to cell. This makes it easier for depolarization and repolarization in the myocardium. Because of these junctions and bridges the heart muscle is able to act as a single coordinated unit.
Cardiac action potential consists of two cycles, a rest phase and an active phase. These two phases are commonly understood as systole and diastole. The rest phase is considered polarized. The resting potential during this phase of the beat separates the ions such as sodium, potassium and calcium. Myocardial cells possess the property of automaticity or spontaneous depolarization. This is the direct result of a membrane which allows sodium ions to slowly enter the cell until the threshold is reached for depolarization. Calcium ions follow and extend the depolarization even further. Once calcium stops moving inward, potassium ions move out slowly to produce repolarization. The very slow repolarization of the CMC membrane is responsible for the long refractory period.
Myocardial infarction, commonly known as a heart attack, occurs when the heart’s supplementary blood vessels are obstructed by an unstable build-up of white blood cells, cholesterol, and fat. With no blood flow, the cells die, causing whole portions of cardiac tissue to die. Once these tissues are lost, they cannot be replaced, thus causing permanent damage. Current research indicates, however, that it may be possible to repair damaged cardiac tissue with stem cells, as human embryonic stem cells can differentiate into cardiomyocytes under appropriate conditions.
Humans are born with a set number of heart muscle cells, or cardiomyocytes, which increase in size as our heart grows larger during childhood development. Recent evidence suggests that cardiomyocytes are actually slowly turned over as we age, but that less than 50% of the cardiomyocytes we are born with are replaced during a normal life span. The growth of individual cardiomyocytes not only occurs during normal heart development, it also occurs in response to extensive exercise (athletic heart syndrome), heart disease, or heart muscle injury such as after a myocardial infarction. A healthy adult cardiomyocyte has a cylindrical shape that is approximately 100m long and 10-25m in diameter. Cardiomyocyte hypertrophy occurs through sarcomerogenesis, the creation of new sarcomere units in the cell. During heart volume overload, cardiomyocytes grow through eccentric hypertrophy. The cardiomyocytes extend lengthwise but have the same diameter, resulting in ventricular dilation. During heart pressure overload, cardiomyocytes grow through concentric hypertrophy. The cardiomyocytes grow larger in diameter but have the same length, resulting in heart wall thickening.
About 128 million people suffer from diseases that might be cured or treated through stem cell therapy. About 58 million of these people suffer from cardiovascular disease.
Cardiovascular disease can manifest itself in many different ways because the blood vessels transport blood to every single part of the body. The heart is the organ that pumps the blood around the body, and it also receives nutrients from the blood vessels (via the coronary vessels). Any interruption of the supply of blood containing nutrients and oxygen to one of the bodys organs leads to functional impairment and, in the worst case scenario, the death of the tissue. One typical example is cardiac arrest, which occurs when the blood supply to the heart muscle is restricted.
Cardiovascular disease can have any number of causes. Some people are born with a susceptibility to vascular disease (e.g. varicose veins), which can be alleviated by taking medication. Other peoples heart and blood vessels can be damaged by external factors. The majority of vascular diseases these days, however, are caused by our modern-day lifestyles. The walls of the blood vessel are always in contact with the blood which flows through them, so they are most commonly affected by unhealthy lifestyles. If someone has an unfavorable haemogram, i.e. if their blood contains too much glucose, cholesterol, triglycerides (fats) or nicotine, this can put the blood vessels under an enormous amount of stress. Glucose adheres to the walls of the blood vessels and the blood constituents, and cholesterol and triglycerides also accumulate on the blood vessel walls. As a result, the blood clumps, the blood vessel walls calcify, turning porous and can no longer perform their biological function properly. Nicotine also constricts the blood vessels, so they narrow and the amount of blood circulating the body is reduced.
If the condition is aggravated by a lack of vessel-protecting substances, the damaged vessels lose their ability to regenerate. The consequences include arteriosclerosis, leg ulceration, dilation of the abdominal artery (aneurysm), cardiac insufficiency, cardiac arrest and stroke. Cardiovascular disease is still the number one cause of death in many other western industrial nations.
Adult Stem Cells derived from the patients own blood are potent and effective to treat heart disease.Patients who have severe cardiac disease with a history of coronary infarction, congestive heart failure, those with previous bypass surgery and stents, cardiomyopathy, and individuals with low ejection fraction (the rate at which the heart pumps) are candidates for this procedure. Patients that survive myocardial infarction have diminished cardiac reserve putting them at risk for subsequent heart failure. Doctors and scientists throughout the world now understand that myocardial repair and regeneration are possible and attainable.
Watch this informative video that highlights one of our patients and how their heart was treated with Stem Cell Therapy. Stem Cell Treatment for the heart saved this patients life.
Regenocyte generates healthy heart muscle cells in the laboratory and then transplants those cells into patients with chronic heart disease. Stem cells cultivated from the patients own blood and transplanted into a damaged heart, can generate new collateral vessels.
Treatment is non-invasive consisting of an intravenous infusion of precursor cardiomyocyte stem cells derived from a patients own blood through a specially designed catheter. This approach increases the engraftment, survival and proliferation of the stem cells to the heart muscle.
To find out more about how stem cells can treat the heart,click hereor call us at (866) 216-5710
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Regenocyte, Stem Cells Used To Treat Cardiovascular Disease
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Challenges in identifying the best source of stem cells …
Endogenous cardiac stem cells (eCSCs) are tissue-specific stem progenitor cells harboured within the adult mammalian heart.
They were first discovered in 2003 by Bernardo Nadal-Ginard, Piero Anversa and colleagues  in the adult rat heart and since then have been identified and isolated from mouse, dog, porcine and human hearts.
The adult heart was previously thought to be a post mitotic organ without any regenerative capability. The identification of eCSCs has provided an explanation for the hitherto unexplained existence of a subpopulation of immature cycling myocytes in the adult myocardium. Indeed, recent evidence from a genetic fate-mapping study established that stem cells replenish adult mammalian cardiomyocytes lost by cardiac wear and tear and injury throughout the adult life. Moreover, it is now accepted that myocyte death and myocyte renewal are the two sides of the proverbial coin of cardiac homeostasis in which the eCSCs play a central role. These findings produced a paradigm shift in cardiac biology and opened new opportunities and approaches for future treatment of cardiac diseases by placing the heart squarely amongst other organs with regenerative potential such as the liver, skin, muscle, CNS. However, they have not changed the well-established fact that the working myocardium is mainly constituted of terminally differentiated contractile myocytes. This fact does not exclude, but is it fully compatible with the heart being endowed with a robust intrinsic regenerative capacity which resides in the presence of the eCSCs throughout the individual lifespan.
Briefly, eCSCs have been first identified through the expression of c-kit, the receptor of the stem cell factor and the absence of common hematopoietic markers, like CD45. Afterwards, different membrane markers (Sca-1, Abcg-2, Flk-1) and transcription factors (Isl-1, Nkx2.5, GATA4) have been employed to identify and characterize these cells in the embryonic and adult life. eCSCs are clonogenic, self renewing and multipotent in vitro and in vivo, capable of generating the 3 major cell types of the myocardium: myocytes, smooth muscle and endothelial vascular cells. They express several markers of stemness (i.e. Oct3/4, Bmi-1, Nanog) and have significant regenerative potential in vivo. When cloned in suspension they form cardiospheres, which when cultured in a myogenic differentiation medium, attach and differentiate into beating cardiomyocytes.
In 2012, it was proposed that Isl-1 is not a marker for endogenous cardiac stem cells. That same year, a different group demonstrated that Isl-1 is not restricted to second heart field progenitors in the developing heart, but also labels cardiac neural crest. It has also been reported that Flk-1 is not a specific marker for endogenous and mouse ESC-derived Isl1+ CPCs. While some eCSC discoveries have been brought into question, there has been success with other membrane markers. For instance, it was demonstrated that the combination of Flt1+/Flt4+ membrane markers identifies an Isl1+/Nkx2.5+ cell population in the developing heart. It was also shown that endogenous Flt1+/Flt4+ cells could be expanded in vitro and displayed trilineage differentiation potential. Flt1+/Flt4+ CPCs derived from iPSCs were shown to engraft into the adult myocardium and robustly differentiate into cardiomyocytes with phenotypic and electrophysiologic characteristics of adult cardiomyocytes.
With the myocardium now recognized as a tissue with limited regenerating potential, harbouring eCSCs that can be isolated and amplified in vitro  for regenerative protocols of cell transplantation or stimulated to replicate and differentiate in situ in response to growth factors, it has become reasonable to exploit this endogenous regenerative potential to replace lost/damaged cardiac muscle with autologous functional myocardium.
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Endogenous cardiac stem cell – Wikipedia, the free …
An interview with Roberto Bolli, MD.
University of Louisville cardiologist Roberto Bolli, MD, led the stem cell study that tested using patients’ own heart stem cells to help their hearts recover from heart failure. Though that trial was preliminary, the results look promising — and may one day lead to a cure for heart failure.
Here, Bolli talks about what this work means and when it might become an option for patients.
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“Realistically, this will not come… for another three or four years, at least,” Bolli says. “It may be longer, depending on the results of the next trial, of course.”
Larger studies are needed to confirm the procedure’s safety and effectiveness. If those succeed, it could be “the biggest advance in cardiovascular medicine in my lifetime,” Bolli says.
A total of 20 patients took part in the initial study.
All of them experienced significant improvement in their heart failure and now function better in daily life, according to Bolli. “The patients can do more, there’s more ability to exercise, and the quality of life improves markedly,” Bolli says.
Bolli’s team published its findings on how the patients were doing one year after stem cell treatment in November 2011 in the Lancet, a British medical journal.
Each patient was infused with about 1 million of his or her own cardiac stem cells, which could eventually produce an estimated 4 trillion new cardiac cells, Bolli says. His team plans to follow each patient for two years after their stem cell procedure.
Keep in mind that this was a phase I study. Those focus on safety more than effectiveness.
The results were “much more striking” than past stem cell trials to heal the heart, Bolli says.
This trial was the first in the world to use stem cells derived from the heart. Earlier studies used stem cells gleaned from different bodily sources, including bone marrow, adipose (fat) tissue, and circulating blood. Those showed either no improvement or only modest gains in a patient’s left ventricular ejection fraction, a measure of the heart’s pumping ability.
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Heart Stem Cell Trial: Interview With Researcher Roberto …