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Back to the laboratory bench

Stem cell therapy for heart problems, which has been tested in humans since the 2000s, has not shown conclusive results and is undergoing new tests in vitro and in animal models

LÉO RAMOS CHAVES Thiago Turaça, a researcher with the InCor team, prepares stem cells for experimentsLÉO RAMOS CHAVES

It was long believed that stem cells extracted from bone marrow or the layer of fat under the skin would solve a long-standing problem in cardiology; these cells can multiply and generate cells that comprise other tissues, and were expected to supply the heart with new cardiac cells. Experiments over the past decade involving humans and animals suggested that they just needed to be implanted in the right place and quantity to halt the natural limitations of this organ, which beats 86,400 times per day and does not replace the cells within it which die throughout a lifetime. Like all mature cells which specialize in a certain function (cardiac cells contract and relax cyclically), these cells have a limited capacity for division, which impedes repair to the organ following events when groups of cells die, such as during a heart attack.

Today we know that adult stem cells are versatile, but not magical. On their own they do not restore the heart, but they do release compounds that can prevent the death of cardiac cells and stimulate the formation of blood vessels. After a period of excessive optimism, specialists at the world’s major cardiology research and treatment centers had to return to their lab benches and perform more tests (involving cells, rodents, and larger animals) while simultaneously continuing to assess the use of these cells in people with heart problems. This necessary strategic retreat may lead to new uses for adult stem cells, which are found in certain tissues of the body but are less versatile than those extracted from embryos; embryonic stem cells can generate any type of tissue. Instead of making weakened hearts work like new, stem cells may optimize the performance of available therapies and medications. The current hopes for repairing cardiac tissue depend on more versatile cells, such as those obtained from induced pluripotent stem cells (see table).

“Today we see adult stem cells in a different way, with limited plasticity [the ability to generate different tissue cells] but still with the potential to modify the environment in which they are introduced,” states biologist Rafael Dariolli, member of a team at the Heart Institute (InCor) of the University of São Paulo (USP) which is investigating the usefulness of stem cells in treating heart problems. The group is led by physician and researcher José Eduardo Krieger, who is leading a clinical trial with the Brazilian Ministry of Health that is assessing stem cells’ ability to improve circulation in the cardiac muscle in people who have chronic ischemia (reduced blood flow to the heart). Alongside the human tests, Krieger and his team have returned to laboratory experiments several times to understand how these cells behave. In one recent study involving pigs which was published in PLOS ONE, Dariolli, Krieger, and their colleagues showed that stem cells extracted from fat increased the formation of blood vessels and blood flow to cardiac tissue, and also reduced scarring caused by heart attacks, helping the heart contract better.

“Science has its own dynamics, which often create uncertainties,” ponders sociologist Maria Conceição da Costa, a professor at the University of Campinas (UNICAMP); she studies how governments, researchers, development agencies, and populations influence national scientific agendas, and is starting to compare the regulations for research and therapies involving stem cells in Brazil and India. “Sometimes more time is needed to understand whether and how certain techniques work.” Krieger agrees: “If a technology is not ready to become a treatment, you need to take a step back.”

If even today no one clearly understands how these cells work, was it hasty to test them in humans in the early 2000s? “I do not believe that the trials started too early,” researcher Enca Martin Rendon of the Oxford Stem Cell Institute, one of the centers linked to the University of Oxford in England, told Pesquisa FAPESP. “We already had extensive knowledge about stem cells from bone marrow, which had been used for more than 40 years in bone marrow transplants to treat some blood cancers.”

RAFAEL DARIOLLI/INCOR-USP A cluster of stem cells from fat tissue grown in the InCor laboratory for implantation into the hearts of pigs after heart attacksRAFAEL DARIOLLI/INCOR-USP

It wasn’t just previous experience with marrow transplants that permitted a rapid transition (over just two years) from animal testing to clinical trials in humans to verify the effectiveness of stem cells in cardiac repair. At the turn of the millennium, there were many studies suggesting properties for adult stem cells which had not yet been imagined. “Since 1999, we have been inundated by high-impact publications indicating that these cells have a plasticity comparable to embryonic stem cells,” recalls physiologist Antonio Carlos Campos de Carvalho of the Federal University of Rio de Janeiro (UFRJ). Along with the Croatian biologist Raduan Borojevic, also a professor at UFRJ, Carvalho concluded experiments in 2001 which suggested that stem cells injected into the hearts of rats with heart failure improved the organ’s ability to beat. This work helped support one of the first Brazilian clinical trials using this type of cell, which concluded in 2003; shortly after, as coordinator of teaching and research at the National Institute of Cardiology in Rio de Janeiro, Carvalho headed one of the largest trials in the world using stem cells for cardiac treatment.

The study that most impacted and stimulated stem cell research in cardiology, the area of health in which studies using these cells have advanced the most, was published on April 5, 2001 in the journal Nature. In a four-page article, a group led by the Italian-American physician Piero Anversa (who at that time was an eminent researcher in the area of cardiovascular diseases at the New York Medical College) announced results that left some cardiac repair researchers astounded and others suspicious. The experiments found that adult stem cells from bone marrow injected into the hearts of mice generated blood vessels as well as cardiac cells that repopulated 70% of the area damaged during a heart attack. More importantly, the new cells appeared capable of contracting and improving the heartbeat. “It was everything we wanted to hear,” says Krieger. “At that time, we believed that the cardiac repair problems were solved.”

There was more. The following year, Anversa and his team reported in the New England Journal of Medicine that in transplanted hearts, up to 10% of cardiac cells originated from the recipient, suggesting that the organ could regenerate from stem cells taken from the circulatory system. Finally, in 2003 the group reported in Cell that they had identified specific stem cells for the heart.

Shortly before that time, some signals had appeared that it was safe to inject these cells into the human heart. In September 2001, the surgeon Kimikazu Hamano and other researchers from Yamaguchi University in Japan reported the results of a clinical safety trial in the Japanese Circulation Journal; this type of trial assesses whether a treatment is hazardous to human health. After testing in dogs, they injected bone marrow stem cells into the hearts of five people who underwent cardiac revascularization surgery. A year later, there were no signs of side effects, and local circulation had improved in three patients. In October of the following year, the cardiologist Bodo Strauer’s group at the University of Dusseldorf in Germany presented data in the journal Circulation which indicated a greater reduction in the area affected by heart attack in the hearts of 10 people treated with drugs and stem cells than in another 10 who received only medication.

Brazil was not far behind. The cardiologist Hans Dohmann and his team at the Hospital Pró-Cardíaco in Rio de Janeiro, in partnership with Emerson Perin, a Brazilian cardiologist at the Texas Heart Institute, had already implanted stem cells into the hearts of 14 patients with chronic heart failure in a study with 21 participants (see Pesquisa FAPESP, issue No. 88). Of the 14 patients who received stem cell treatment, 12 survived and showed increased circulation and contraction of the heart, according to data published in Circulation in 2003. No significant improvements were seen in the individuals in the control group.

RAFAEL DARIOLLI AND CHRYSTIAN J. ALVES In green, cells resembling heart cells which were derived from pluripotent cellsRAFAEL DARIOLLI AND CHRYSTIAN J. ALVES

But around this time, doubts began to emerge. Various groups intrigued by Anversa’s findings attempted to repeat his experiments, without success. Charles Murry at the University of Washington and Loren Field at the University of Indiana published an article in Nature in 2004 stating that in contrast with what Anversa had found, bone marrow stem cells did not transform into cardiac cells. Suspicions around Anversa’s work grew over the following years and led Harvard University, where the researcher had later transferred, to open an investigation into some studies, requesting the withdrawal of one article from 2012 and calling another from 2011 into question; his previous findings were never reproduced, causing a loss in credibility. “Knowledge in this area worked like a pendulum,” says Carvalho of UFRJ. “It was initially believed that adult stem cells gave rise to anything, [and then] years later, the most important scientific journals were publishing articles indicating that none of this happened.”

These setbacks did little to thwart the generalized optimism. The tests in progress were generally small (phase 1), intended to confirm treatment safety. Like the trial conducted by Dohmann in Brazil, they nearly always showed encouraging results. At that time, phase 2 studies with more participants and more rigorous methodology were beginning; these trials compared injection of stem cells with that of a harmless compound (a placebo) and randomly selected which would be administered to each participant.

As the results of the phase 2 tests came in, the initial magic of stem cells began to fade. The POSEIDON study, which involved 37 people in the United States, the BOOST-2 study, with 153 patients in Germany and Norway, and the TECAM Trial, which involved 120 participants in Spain, showed more modest benefits than previous results.

In Brazil, previously published data from one of the largest clinical trials assessing the effectiveness of stem cells in cardiac repair are also not encouraging. This multicenter randomized study on cellular therapy in heart disease (Miheart) was launched in 2004 by the Brazilian Ministry of Health; it cost R$13 million and studied the treatment of 1,200 patients with heart attack, coronary artery disease, dilated cardiomyopathy, and damage to the heart caused by Chagas disease using bone marrow stem cells.

Two of the study’s four arms have already been completed, and the results were published without much fanfare. One of the arms of the Miheart study evaluated injection of stem cells into the arteries that feed the heart to improve cardiac function in 183 people with Chagas disease. Another investigated the effect of these cells in 160 participants with dilated cardiomyopathy, when the heart enlarges and has difficulty pumping blood. In both cases, the improvement caused by the cells was similar to that seen in patients who received the placebo. The data from the third arm (heart attack) have been submitted for publication and also do not show advantages; data from the fourth arm (coronary artery disease) are currently under analysis. From what is known so far, there is no reason to indicate the use of these cells to treat the problems assessed in the Miheart trial, at least not for the generally severe level of the cases included in the study.

Meta-analyses, studies which use statistical tools to group similar clinical trials and increase the number of cases to identify more subtle effects, have been equally inconclusive. One of the reasons is that the trial designs vary, making it difficult to analyze the data together; for example, the strategy for injecting stem cells may differ (into the arteries or into the cardiac muscle, for example) and the measured outcome can also change (such as increased pumping capacity or reduced mortality). In a commentary published in June of this year in the British Medical Journal, Enca Martin Rendon and his group stated, after analyzing 38 trials, that results from studies with higher numbers of participants are necessary before the use of adult stem cells can be recommended in clinical practice.

One of the studies they are awaiting is the European BAMI clinical trial, which assesses the use of bone marrow stem cells in three thousand heart attack victims. “If BAMI does not show benefits, there will be nothing more to discuss,” Carvalho says. “We will need to move on to other types of cells.” Despite this critical view, he has not given up. Carvalho returned to animal tests, and along with parasitologist Maria Terezinha Bahia of the Federal University of Ouro Preto (UFOP) he is attempting to use stem cells from fat tissue (which are more homogeneous than bone marrow stem cells) to treat the hearts of dogs with Chagas disease.

At InCor, Krieger is still optimistic. In his view, the experiment in pigs and the revised role of adult stem cells open new possibilities for use, particularly for stem cells extracted from fat. He and Dariolli attribute the improvement they saw in the pigs to these cells’ capacity to make the area surrounding the injury less hostile. The cells release at least 30 compounds; some stimulate the formation of blood vessels, while others may control inflammation and avoid greater deterioration of blood-starved tissue. “These cells do not repair the heart, but may increase the effectiveness of existing clinical and surgical treatments,” says Krieger. He also suspects that they may benefit people with less severe heart damage than those who were treated in the early clinical trials. Enca Rendon of Oxford also sees potential in these cells—and in immature heart cells (cardiac progenitor cells)—to alleviate heart failure, which currently is treated by transplantation of the organ.

Tests in other countries have already begun using stem cells which appear to be more versatile. The cardiologist Eduardo Marbán and his team at the Cedars-Sinai Heart Institute in California have already inserted cells obtained from cardiac progenitors and subsequently cultured in the laboratory into human and animal hearts. A safety trial involving 17 heart attack patients found that the procedure was safe and reduced scarring in the heart, according to a 2014 article in the Journal of the American College of Cardiology.

At the University of Osaka, Japan, cardiologist Yoshiki Sawa and his colleagues implanted human cardiac cells (cardiomyocytes) derived from induced pluripotency stem cells (iPS) into pig hearts; iPS are adult cells which have been reprogrammed to behave like stem cells. Initial analyses published in August of this year in Scientific Reports indicate that the cells incorporated themselves into the heart and improved its ability to pump blood. But the results are not always so encouraging. At the Symposium on Cardiovascular Regenerative Medicine, which was held in September in the United States, Michael Laflamme of the Toronto General Hospital Research Institute in Canada presented the results of his experiments using human cardiomyocytes obtained from iPS cells. When these were implanted into pig hearts, they integrated themselves into the region affected by the heart attack, but no benefits were seen; furthermore, of the seven animals who received this treatment, two died as a result of changes in the heartbeat (arrhythmia). “Now they are trying to understand what causes this arrhythmia,” says Carvalho, who participated in the symposium.

“In medical science, there is more controversy than certainties,” says Rafaela Zorzanelli, professor at Rio de Janeiro State University (UERJ). She and her colleagues traced the evolution of stem cell research in Brazil in an October 2016 article in the journal História, Ciências, Saúde – Manguinhos. “Today perhaps there are less grandiose expectations about the use of stem cells. I do not believe that it is a step backwards, but a more realistic view of what to expect from this type of biotechnology.”

Project
Cardiovascular genomics: Mechanisms and new therapies – CVGen mech2ther (No. 13/17368-0); Grant Mechanism Thematic Project; Principal Investigator José Eduardo Krieger (InCor-USP); Investment R$6,902,193.63.

Scientific article
DARIOLLI, R. et al. Allogeneic pASC transplantation in humanized pigs attenuates cardiac remodeling post-myocardial infarction. PLOS ONE. Apr. 27, 2017.