The Use of Stem Cell Therapy for Cardiovascular Disease

Stem cell therapy is now being applied clinically to the heart and is well out of the preclinical realm.

Overview

In the overall world of cell therapy, we have skeletal myoblasts on one side, and bone marrow cells on the other. In general, when we talk about myoblast therapy, we're really talking about administering committed muscle cells, as I'll discuss shortly. On the other hand, when we talk about bone marrow cell therapy, we're talking about a stimulus for angiogenesis, be that through transdifferentiation, or fusion, or some type of paracrine effect.

Bone marrow stem cells are an autologous product. Since they are adult cells, there are none of the potential moral or ethical issues that surround fetal stem cells. They can be introduced using transcatheter or surgical approaches. For instance, we are currently applying a transcatheter injection approach in patients with chronic heart failure. You can also infuse autologous bone marrow cells in the coronary system. This approach has been tried in a number of trials in patients with acute myocardial infarction, one of which was recently published, and another of which is ongoing in Europe right now.

Another approach is an indirect approach giving granulocyte colony stimulating factor (GCSF), which stimulates cells to come out of the bone marrow into the circulation. You get a very high white count, but more importantly, more circulating progenitor cells. If there is an ongoing inflammatory process, associated, for example, with a recent myocardial infarction, the theory is that these cells will go to the area of injury and help heal and improve function. So far, this approach alone has been somewhat disappointing; but this drug has also been used to assist in the harvesting of bone marrow cell. GCSF makes it easier to get larger numbers of certain specific types of cells for application in stem cell therapy.

Myoblasts

Myoblasts are derived from satellite cells that are cultured; they are actually an early committed muscle cell and not really a true stem cell. Nonetheless, this approach is also currently under investigation. It involves taking a small specimen from a skeletal muscle, such as the thigh; the cells are then cultured over a period of time. These cultured cells are then implanted directly into the injured myocardium, either via catheter or surgically. Although there have been a few small studies, usually associated with other kinds of revascularization or with left ventricular assist devices, there now is a large multicenter randomized trial ongoing in Europe, and another investigational new drug protocol here in the United States using transendocardial injection of myoblasts.

Although the implanted myoblasts may not develop a normal connection with the surrounding muscle, they may have a functional role when implanted. Studies have shown objective evidence of improvement in perfusion and function (such as ejection fraction), but the major drawback of this kind of therapy is that these cells are electrically active. There have been cases of sudden death, so much so that in protocols in which myoblasts are implanted in the heart, an automatic implantable cardioverter-defibrillator is also routinely implanted.

Bone Marrow Stem Cells

Turning now to bone marrow cell therapy, this is an area that I personally have been very involved with, along with my colleagues at the Texas Heart Institute. There are many different cellular types in the bone marrow. The cells that are part of the stroma are the mesenchymal cells that have the potential to turn into almost any tissue in the body. There are other cells that are progenitor cells for vascular structures, and you can easily imagine how they might come in handy. And then you have precursor cells for the hematopoietic cell line. When you do a bone marrow aspiration, you're really getting a lot of different cells; a key question is which specific cell type might be particularly beneficial for which disease state.

You can use unselected mononuclear cells as sort of a general approach, or you can select certain types of cells, such as endothelial progenitor cells that have a CD34 marker or—even one step more primitive AC133 expression. You can take mesenchymal cells directly from the bone marrow, or you can stimulate them to come out of the bone marrow and then collect them peripherally. There are even stem cells in many different tissues; you can harvest fat and obtain stem cells. Other sources include placenta or blood; so many different approaches are possible.

Angiogenesis

Over the last few decades, our view of angiogenesis has changed. The initial belief was that blood vessels were mostly created in the embryonic phase of life, but we learned that angiogenesis occurred in adults, as well, in things like tumors. Then, in the late 1990s we learned that adults have circulating cells that can form blood vessels. A lot of attention has focused on ischemic heart disease as a model of impaired vascularization, but this paradigm can also be applied to other types of cardiac disease. Dr. Anversa and his group at Columbia have changed the notion of the heart as an end-stage organ; they postulate that the heart may actually renew itself, and there may be resident cardiac stem cells in the heart, which perhaps can be stimulated. This might be one of the targets affected, for example, by the paracrine effects of exogenously administered bone marrow stem cells.

Potential paracrine effects may be very important. One of these progenitor cells may be secreting 10 different growth factors, another 10 different cytokines, and chemokines and other proteins. Such a cell has an ability to secrete an incredibly complex cocktail of proteins that can be beneficial, depending on the milieu in which they're placed.

Let me give you a very quick crash course in immunofluorescence histopathology, a clever way of marking cells to elucidate the biology. FITC is a green dye label; you can attach it to antibodies that will go to endothelium, antibodies that will go to smooth muscle, and antibodies that will go to cardiomyocytes. So we have a way of making the endothelium of a particular animal light up in green. Now suppose that the cells that we're injecting into these animals are labeled with Di I, which is a red dye. If you co-localize green and red, you obtain a yellow structure. So if you see a yellow structure, you know that structure was made from cells that were injected into the body and co-localized. We have shown exactly this phenomenon in the basic laboratory: endothelium of vessels created by co-localized cells injected into the animal, not there originally (Fig. 1).

Fig. 1 In this immunofluorescent histopathologic study, antibodies that travel to the endothelium have been labeled with FITC green, and stem cells injected into the canine model have been labeled with Di I red. The yellow areas, evident at 4 months after the procedure, are new endo-thelium marked by the co-localization of red and green.

We have shown the same co-localization in the smooth muscle cell layer of this vessel. So exogenously administered cells helped create both endothelium and smooth muscle cells, the major components of arteries.

Delivery

There are a couple of delivery approaches. You can increase endogenous production and hope that the cells target the injured area, but I've already suggested that this has limited effectiveness. More commonly, we want to somehow inject them into the injured tissues. Intravenous injection is not very efficient. You lose the vast majority of the cells in the lungs, spleen, and lymphatic tissues, and you really get a very, very low efficiency of delivery. You can deliver it intra-arterially; but while that may be feasible in an artery that you have just opened up and can pour cells down, this is much more difficult in the chronic heart failure patient who may have concomitant critical coronary disease, or even occluded vessels. How can you be sure to get it where you need it? And, finally, you can use a surgical approach and direct injection if you have an open chest.

Our preferred method of delivery is actually a hybrid of these: catheter-based transendocardial cell delivery. This can be guided with fluoroscopy (which is not very precise), with intracardiac ultrasound (which is improving but is not really there yet, in regard to accessing all areas of the heart), with MRI (which is not yet practical for this application), or with electromechanical mapping, which is our favored technique. Once we have mapped the area to be treated in the lab, we can very precisely guide the transendocardial injection of cells to the target area with a catheter-directed needle. With special stains, we have been able to show in animals that new cells injected into the border zone actually move to the area of the infarct.

Clinical Experience to Date

The BOOST trial, published recently, is the only randomized trial of intracoronary stem cell therapy in acute MI patients to date. It involved 60 patients, and treatment (autologous bone marrow cells versus control) was administered approximately 5 days after a heart attack; the follow-up period was 6 months. At 6 months, there was a mild increase in ejection fraction in the stem cell group, from about 49% to about 53%. But this has generated a lot of interest, and a similar large randomized trial—the REPAIR AMI—is ongoing.

A few years ago, we did a study in Brazil, which was the first human application of mononuclear cells (transendocardially delivered) in heart failure patients. We treated 14 patients in a trial with 7 controls, chiefly in order to demonstrate procedural safety; we've also published 6- and 12-month clinical follow-up that showed dramatic improvement in a number of cases. From there, we talked at length with the FDA, and we were granted approval to initiate our own IND of this approach in heart failure patients who have some evidence of reversible ischemia. Our Texas Heart Institute trial includes 30 patients—20 treated and 10 controls—again with a primary endpoint of safety. We are currently midway through enrollment, having just enrolled patient number 15. I cannot share our results at this time, because the trial is ongoing, but this will be a pivotal study for the future application of this technique.

Finally, I want to present histological data from one of the patients who was treated with autologous mononuclear cells in our initial study in Brazil. This patient died 11 months out, and we were able to obtain specimens for histologic analysis. The anterolateral wall of the heart was the area that was injected, and when you compare trichrome stains of the injected area to the normal area, you see a substantial increase in vascular density in the injected area. There was also very prominent staining for filament precursors (such as desmin) in developing cells.

Summary

As I've shown today, stem cell therapy is not just theoretical—it is now being applied clinically for cardiac disease. There will be data forthcoming on intracoronary injection in acute MI and our own study of transendocardial injection in heart failure. We still have a lot to learn about the biology, but everything that we are learning just makes this field more exciting.