Animal/in vitro/Clinical study with bone marrow and heart attacks

An interesting paper was published in Asian Cardiovascular and Thoracic AnnalsSoma Guhathakurta, Usha R Subramanyan, Ramesh Balasundari, Chandan K Das, Nainar Madhusankar, and Kotturathu Mammen Cherian, Stem Cell Experiments and Initial Clinical Trial of Cellular Cardiomyoplasty Asian Cardiovasc Thorac Ann, Dec 2009; 17: 581 – 586.

In this paper, several Indian scientists used bone marrow cells to test three possibilities:  1) to determine whether transplantation of a sheep’s own bone marrow stem cells into the infarcted myocardium could promote the differentiation of those bone marrow cells into beating heart muscles; 2) demonstrate that bone marrow mesenchymal cells could differentiate into heart muscle cells; 3) conduct a clinical trial with patient’s own bone marrow to determine if it could improve the heart function of patients with severe heart problems.

The first stage used five experimental sheep in which a heart attack had been induced (coronary artery ligation), and two control sheep (they started with nine, but one control animal and one experimental animal died).  The control sheep did not show any improvement, but the experimental sheep, which received 100,000 to 1,000,000 bone marrow mononuclear cells (injected into the periphery of the infarcted area), showed improvements in blood circulation throughout the heart.  Echocardiography demonstrated marginal improvements in ejection fraction at 3 months post-injection.  Staining of the heart tissue from the injection site revealed that some of the heart tissue that should have died was still alive.  An even more remarkable finding was that throughout the scar, islands of muscle tissue that could not be differentiated from the
surrounding muscle tissue.  Since four of the experimental sheep had been injected with unmarked bone marrow, this large island of heart muscle tissue in the heart scar had a different look from the rest of the surround heart muscle.

One experimental animal, however, had been infected with a virus that causes the bone marrow to glow (Green Fluorescent Protein).  When this experimental animal was examined with UV light, the island of heart muscle tissue in the scar glowed, demonstrating that the new heart muscle is from the implanted bone marrow.

In the second stage of their experiment, the researchers tried to grow bone marrow cells in culture and differentiate them into heart muscle cells.  The technique they used is apparently undergoing a patent process.  Therefore, they did not reveal the technique.  Nevertheless, the bone marrow cells formed structures that looked like heart muscle, expressed many heart muscle genes (GATA-4, Nkx 2.5, hANP, MLC-2v and MLC-2a), and in the electron microscope, had a definite heart muscle-like structure, but without the structures that connect heart muscle cells together (intercalated discs).

A marginal improvement in myocardial function was noted at 3 months (mean increase in ejection fraction, 6% ±1%), although it plateaued at 6 months. The trial proved tobe safe because there was no procedure-related mortality. There is growing optimism that stem cell therapy may delay heart transplantation.

Finally, they transplanted bone marrow cells into the hearts of 29 patients who suffered from “dilated cardiomyopathy,” which simply means that they have an enlarged heart, or “endstage ischemic cardiomyopathy,” in which the blood vessels of the heart are so clogged that the patient’s heart cannot receive adequate amounts of oxygen even at rest.  Also 11 other patients with similar heart problems were injects with endothelial progenitor cells (EPCs), which are also found in bone marrow, but make blood vessels, and occasionally heart muscle.  One particular patient was 5-months old and had a congenital case of cardiomyopathy.  In this patients, EPC injections caused a spike in the ejection fraction from  32% to 58%.  Other EPC-injected patients produced mixed results,from no improvement to a 7% increase in ejection fraction, with no mortality.  The differences in the results is probably due to the fact that they used three different ways to deliver these stem cells; intracoronary delivery, direct injection into the heart muscle, and infusion through the pulmonary arteries.  A marginal improvement in heart function was noted at 3 months (mean increase in ejection fraction, 6% ±1%), although it plateaued at 6 months.  The trial proved to be safe because there was no procedure-related mortality.

This study is a nice example of the combination of animals, test-tube, and clinical studies.  The problem is that the clinical study is too unfocused to properly interpret.  It is small, and non-randomized.  There is no placebo, and the patients showed a wide variety of chronic heart conditions.  The fact that no one became horribly ill because of the procedure is encouraging, but the results are all over the board, as are the patients and their conditions, which makes the clinical trial inconclusive with respect to efficacy.  This study definitely justifies a larger, more focused, randomized study.  Also, the animal and in vitro portion of the study gives excellent, positive evidence that mesenchymal stem cells from bone marrow can transdifferentiate into heart muscle cells.  However, the in vitro work shows that the heart muscle made by the mesenchymal stem cells did not make connexion-43, which is essential for gap junctions and physiological connection between the heart muscle cells.  This lack of physiological integration can lead to functional isolation of the new heart muscle tissue, which can generate arrhythmias.  Engineering these cells with Connexin-43 would be a good start, which has been shown in skeletal muscle cells to prevent functional isolation and arrythymias (see Tolmachov O, et al. Overexpression of connexin 43 using a retroviral vector improves electrical coupling of skeletal myoblasts with cardiac myocytes in vitro. BMC Cardiovasc Disord. 2006 Jun 6;6:25).  The second concern is that these presumptive heart muscle cells were not tested for calcium handling proteins.  This is important, since some publications that shown that bone marrow stems cells can form cells that approximate heart muscle cells, but these cells lack the calcium-handling machinery necessary to generate a heart beat (Scherschel JA, Soonpa MH, Srour EF, Field LJ and Rubart M (2008). Adult bone marrow–derived cells do not acquire functional attributes of cardiomyocytes upon transplantation into peri-infarct myocardium. Mol Ther 16: 1129–1137).  These scientists should go back to look at this as well.

All in all this is a fascinating study that provides some excellent evidence, but also leaves many hanging questions.