Hearts without cells? What is this Madness?

Nature medicine has (or is about to, I can never really tell with Advanced online publishing) come out with the news that a group of scientists have made a breakthrough in regenerative heart medicine

What they have done (in simple terms) is take out the heart of a dead rat, removed all the cells, leaving this ghostly shadow of an organ, and then added a fresh batch of stem cells to the mix, thus creating (as if by magic) a new heart.


However, it’s a more complicated than that (otherwise we could all do it in our backyard sheds) and I’ll try to explain it as best as I can.

What this new finding represents is a new step in developing stem cells for curing heart disorders.
One of the main causes of death in the UK is Heart disease. This “disease” in itself is usually a conglomeration of various different disorders, which are caused by defects in the cells of the heart, or in the vasculature.

What is Heart Disease ?

Cardiomyopathy- Your heart is basically a muscle designed to pump blood around your body. All of the heart cells are essentially muscle cells, each containing a series of muscle fibres (Actin and Myosin), which tug on each other to create a contraction. Cardiomyopathy is a disease where these muscle cells die off, reducing the contractile strength of the heart muscles, and their ability to conduct contraction to the rest of the heart. This disease can lead to arrhythmias, and sudden cardiac death. There are many different cardiomyopathies, which can be caused by environmental factors (restriction in blood flow, immune reactions, drugs) or by genetic factors.
Cardiovascular disease: This is generally caused by any blockage of blood vessels supplying the heart. This can lead to some parts of the heart being oxygen starved, and dying off, which can lead to cardiomyopathy.
Coronary Heart Disease: this is usually caused by a build up of plaques within blood vessel that supply the heart. This disease can cause Angina, and heart attacks.
Hypertensive Heart disease: this is caused by a high blood pressure. This can cause increased growth of the left side of the heart as the muscle tries to cope with the pressure (Left side hypertrophy), death of cells ( Hypertensive cardiomyopathy) and cardiac arrhythmias.
Valvular heart disease: This sort of disease shows up as a heart murmur. These effect the pumping ability of the heart, and can have a number of causes , ranging from the genetic, to the immunological. These can lead to angina, hypertrophy of the heart muscles. The symptoms depend on which valves in the heart are damaged.
Inflammatory Heart Disease: this is where an immune response causes the heart walls to swell up, which can block heart valves. Also, if the muscle is affected, it can lead to a cadiomyopathy, and leads to rapid signs of heart failure, and sudden death.
Heart Failure: This is a catastrophic failure of the heart to fill up with blood, or pump it out of the body, and can result from a variety of disorders, such as the ones described above.

The main problems people get with their heart stem from the death of cardiomyocytes (the heart muscle cells). The heart is not very good at regenerating itself after these cells die off [3]. Often, the heart will simply develop scar tissue, which covers the area where cells have died, but does not replace their vital function. So after you have one heart problem, you will have more and more.

Stem Cells to the Rescue !!

Many of these diseases can be treated with drugs or minor surgery. If there is a blockage of some of the blood vessels, heart bypass surgery can be used to allow the reperfusion of the blood to the heart.
When someone is in the terminal stages of heart failure, when there is widespread cardiomyopathy, the only way to effectively save their lives is to give them a heart transplant. This in itself has a lot of problems. Firstly, you need to find a heart with matching tissue type. This can be quite difficult, as not many people allow themselves to be organ donors (unless Gordon Brown gets his way) and most people who die generally don’t die with perfectly preserved hearts.
So other ways have been sought out to replenish the levels of cardiomyocytes within the heart, in order to reverse previous damage. Research into this field has been going on for over ten years.
In the first attempts, people attempted to graft skeletal muscle cells onto the heart, as heart muscle and skeletal muscle are in many ways similar. They used myoblasts, which are a form of stem cells which are present in the muscle, and whenever damage occurs to muscle, they grow and repair the damage.
The only problem with this method is that while in many ways, skeletal muscle is similar to heart muscle, in many other ways it is quite dissimilar. Heart muscle cells have to perform two main functions. They have to respond to electrical stimulation produced by the pacemaker cells of the heart (usually found in the sino-atrial node and the atrio-ventricular node of the heart) by contracting. Skeletal muscles can perform this function quite well, as skeletal muscles have to respond to electrical signals (provided by the nerves connected to the muscles via the neuromuscular junction)
However, cardiomyocytes also need to conduct the electrical signals to their adjacent cells via gap junctions. Skeletal muscles are one of the few cell types that cannot form these junctions, and so cannot conduct electrical stimulation. [2] However, the therapeutic effect of using these cells was indisputable, and clinical trials were carried out in the year 2000.
Ideally, the right cells to use would be cardiomyocytes. However, the Adult stem cells for cardiomyocytes have been difficult to find. Foetal stem cells for the heart have been isolated, and they are able to implant into the hosts heart with little or no trouble [1]. Later experiments showed that foetal cardiomyocytes are perfectly capable of repairing damage inflicted upon the heart [3]
However, the degree of repair that these cells can perform is limited by the massive amounts of necrosis that they encounter when they reach the heart. When necrosis occurs, lots of toxic substances are released, and these can cause a lot of problems for growing stem cells.
At the end of the 20th Century, scientists were beginning to experiment with embryonic stem cells, in order to obtain a new source of cardiomyocytes.
But even more recently, things have taken a strange turn, in that an adult stem cell line, known as mesenchymal stem cells, have shown themselves to be able to differentiate into a whole variety of cells, including cardiomyocytes. I have already waxed lyrical on these cells in a previous post.
There have been trials of mesenchymal stem cells that have showed that as a cell based therapy, these cells can elicit some improvement in patients [4]. However, it should be noted that in studies in Doberman dogs (which have a natural predisposition to heart failure) the administration of mesenchymal stem cells triggered lots of small micro infarctions. So far nothing of this sort has been observed in humans or rats, but it is something to keep an eye out for.

Stem cells in general can help regenerate some of the heart’s function. However, in the case of heart failure, where large portions of the heart are no longer functional, it is nearly impossible to get enough cells to the right location and in high enough concentration to get an effective response.

Because of this problem, research has been done into taking these stem cells, and putting them together so that you can grow a patch of heart muscle which can then be grafted onto an organ. This will work a lot like transplantation, but the stem cells will have to be taken from the individual with the problem, thus avoiding any graft vs. host complications. The dream pursued by people performing this sort of research is that they would some day be able to create an artificial heart to replace the old one.

However, this sort of tissue engineering is harder than it would first appear.

How would you go about growing heart muscle in the lab ?

One of the main problems with doing this is that heart muscle is a complicated tissue, and the heart itself is not a simple tissue. The heart has many different types of cells , which are organised in a specific way. The pericardium has to be on the outside of the heart, to minimise friction with the lungs when it beats. The myocardium has to be thick, and also be well vascularised, to allow the flow of blood into it. Not to mention the different electrophysiological properties of the heart which need to be replicated in order to a regular rhythmic beating motion. You will need to make sure that purkinje cells segregate into the septum of the heart, and that the cells in the atrium don’t beat at the same time as the ventricles (that would bring the heart close to exploding).
This science has quite long roots in the past; the first attempts at growing isolated heart tissue had begun in the 1950’s. These experiments used chick embryo’s to generate artificial heart tissue [5]. After 18 hours in a bioreactor, Moscona found that the embryonic heart cells arranged themselves into a structure similar to that found in the heart. They even had a similar functionality. Cardiomyocytes had a compulsion to aggregate and form tissue, and what’s more this tissue could be observed to start beating rhythmically.
This finding was later expanded upon in the 1980’s, when researchers decided to grow these cardiomyocytes on a collagen membrane. The cardiomyocytes in a normal cell are bound by the extracellular matrix, which provides them with a solid surface to attach to so that they can contract. This matrix is made up mostly of a long stringy molecule known as collagen. When researchers grew their cardiomyocytes on the collagen gel, they found that these cells began more differentiated, and more heart-like.
In later experiments, it was found that the cardiac muscle cells aligned themselves according to the direction of the collagen molecule.

The efficiency of these tissue grafts is dependant on the type of scaffold they are grown on. This scaffold has to be constructed along the lines of the target organ. The tissue you generate has to be as similar as possible.

However, there is a limit to how big a tissue you can make. Heart tissue needs to be heavily vascularised, in order that the cardiomyocytes all get enough oxygen to survive. However, in the lab it is quite difficult to produce tissue with vascular cells within it that can conduct blood, because you will need to link them to the systemic circulation, which is quite difficult. Because of this, there is a critical size for tissue grafts, which is defined by the ability of oxygen to diffuse through that particular tissue.

In order to combat this, there is some research going into impregnating the scaffolds upon which these tissues are built with angiogenic factors within them, so that when the tissue is implanted, blood vessel naturally grow into it, and fill up all the layers with blood. Another technique is to input the cells which will later develop into blood vessels into the construct, in the hope that they will mature into blood vessels.

So where does the De-Cellularised heart fit into this ?

Sometimes, it’s best to go back to nature and see how evolution solved the problem that we are trying to tackle. Instead of trying to construct complex collagen gels impregnated with all sorts of nutrient factors, why not go and see how the actual heart solves this problem?
This was what a group at the University of Minnesota decided to find out [7]. So they decided to do something quite ambitious and remove all the cells from a rat heart, and see what was left over.
They did these using a series of different detergent solutions. Not like fairy liquid, I mean proper detergents, a chemical detergent is designed to mix itself with lipids, but still have an end poking out which is attracted to water. They took the heart out of a dead rat, and then flushed PBS (to clean out the cells) and then SDS detergent under pressure for 12 hours. At the end of this, all of the cells ended up getting washed off of the heart, in theory leaving only the extracellular matrix scaffolds that make up the heart. In order to check that they got rid of all the cells, they looked at the histology of their “ghost hearts” (I’m always slightly sceptical of histological evidence, because what it involves is looking under a microscope at the tissue. It’s easy for even the best scientists to miss something).
But anyway, they looked at a variety of ways to make sure that no cells from the original host were left, and found that they were all gone.

So now they have a de-cellularised heart. What next? What was the point?

Now they have a fantastic scaffold upon which they can build a “new” heart. It should be the ideal scaffold, because it is this structure which tissue engineers have been spending a lot of time trying to mimic, so it should be a good scaffold.

What they did next was stick on foetal cardiomyocytes onto this scaffold.
Before doing this, the heart had to be perfused with the right nutrient medium at the right temperature, and the right pressure, before you could introduce your first cells. These cells then began growing, and 8 days after the cells were injected, the hearts became responsive to electrical stimulation, showing that the cells had grown and re-populated the heart.
Then, endothelial cells were injected (these are the cells that develop into blood vessels) to hopefully improve the growth of cells within the heart. However, the cellularisation was not evenly distributed, it was found to be the best at points where the cells were injected.

So what does this new piece of research leave us with ?

The important thing here is that for the first time, we are now able to look at how the extracellular matrix of the heart is arranged in a clear way. The structure of this natural scaffold will be incredibly for the tissue engineers trying to mimic it. So in this respect it is quite important.
The fact that they were able to re-cellularise the heart is also a big step. This leaves a new gateway open for the organ transplant. Most individuals in the population don’t have the same tissue type. This experiment could potentially show another way for organ transplants to work. Instead of just transplanting the heart from a cadaver, what could be done is the donor heart could be de-cellularised and re-populated with stem cells from the recipient, to create a heart tissue with no tissue incompatibility problems.

However, I would not start goring a huge amount of hamburgers yet, because just because they could do this in rats does not mean that it’s possible in humans.
For a start , a rat heart is really small. Much smaller than a human heart. It’s also not just a smaller version of the human heart either. If the human heart was shrunk to the size of a rat’s, it would still have thicker walls.
The method that was used to de-cellularise these cells is very dependant on size and pressure. If you were to do the same treatment with a human, you would need the put the detergent through it at a higher pressure, and you will have to do it for a lot longer. The human heart has much more cells than a rat’s heart, and to get rid of all the cells would take a lot of time. The risk of using higher pressures and taking more time over this experiment is that there is no guarantee that changing these will not severely alter the structure of the structure of the collagen.
Rat hearts are generally quite different from humans, and even rat cells are very different. The rat heart beats at 300bpm, the electrochemical coupling of these cells is also quite different, and the force frequency relationship between heartbeats is negative (whereas in humans, this relationship is positive). So looking at the structure of the cellular matrix of the rat may be useful, but because of these intrinsic differences between rats and humans, I would not say that the problems of tissue engineering the heart are all solved.
Ideally, this experiment should be replicated in animals which are more similar to humans, such as pigs, or even sheep. But even then, I wouldn’t start celebrating until they had a viable heart transplant with these re-cellularized hearts, and we are a way off that yet.
However, this experiment has opened up a whole new area of research, and I suspect we will see more experiments with de-cellularized hearts popping up next year. Not many, because it is still quite an expensive procedure, which requires a lot of time as well.

1. Soonpaa MH, Koh GY, Klug MG, Field LJ: Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. Science 1994, 264(5155):98-101.
2. Scorsin M, Hagege A, Vilquin J-T, Fiszman M, Marotte F, Samuel J-L, Rappaport L, Schwartz K, Menasche P: COMPARISON OF THE EFFECTS OF FETAL CARDIOMYOCYTE AND SKELETAL MYOBLAST TRANSPLANTATION ON POSTINFARCTION LEFT VENTRICULAR FUNCTION. J Thorac Cardiovasc Surg 2000, 119(6):1169-1175.
3. Caspi O, Gepstein L: Stem cells for myocardial repair. Eur Heart J Suppl 2006, 8(suppl_E):E43-54.
4. Chen SL: Effect on left ventricular function of intracoronary transplantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. The American journal of cardiology 2004, 94(1):92-95.
5. Moscona A, Moscona H. The dissociation and aggregation of cells from organ rudiments of the early chick embryo. J Anat. 1952; 86: 287–301
6. Leor J, Aboulafia-Etzion S, Dar A, Shapiro L, Barbash IM, Battler A, Granot Y, Cohen S Bioengineered cardiac grafts:A new approach to repair the infarcted myocardium? Circulation. 2000; 102: III56–61.
7. Ott HC, Matthiesen TS, Goh S-K, Black LD, Kren SM, Netoff TI, Taylor DA: Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart. Nat Med 2008, advanced online publication.