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Interview

Radovan Borojevic: The tamer of cells

The Croatian-born biologist and UFRJ professor emeritus speaks of the obstacles and encouraging perspectives for regenerative medicine

Borojevic at his home in Petrópolis, where he has been self-isolating since the beginning of the pandemic

Leo Lemos

At nearly 80 years of age, Radovan Borojevic has no complaints about his life. “I have had more opportunities than I was able to take,” says the biologist. “I feel obliged to give back through my work.” The son of a Serbian military man and a Croatian mother, Borojevic was born in Zagreb, in the former Yugoslavia, in November 1940—a week before the country was invaded by Germany and dragged into World War II. He was starving during the conflict and grew up in a socialist dictatorship. Economic and social challenges forced him to leave the university in Zagreb and immigrate to France.

In Strasbourg, he had the chance to finish his studies and, under the guidance of biologist Claude Lévi, he dove into a topic that shaped  his scientific career: the quest to understand how cells communicate and influence their environment, critical for the development of therapies that regenerate injured tissue.

Borojevic moved to Brazil in the late 1960s and, in 1980, was hired by the Federal University of Rio de Janeiro (UFRJ). There, he helped implement the first bone marrow transplant service in the state of Rio de Janeiro and develop pioneering stem cells studies to treat cardiovascular and degenerative diseases. Now, retired from the university, he heads the largest cell bank in Latin America, providing more than 300 cell lines for research, and which he established in the early 1980s. The bank is currently located at the National Institute of Metrology, Standardization, and Industrial Quality (INMETRO).

In the following online interview, which took place in August, he talks about the main challenges and advances in regenerative medicine in Brazil and worldwide.

Age 79
Field of expertise
Cell therapies and tissue regeneration and repair
Institution
Federal University of Rio de Janeiro (UFRJ)
Educational background
Graduate degree in biology from the University of Strasbourg (1963) and PhD from the University of Paris 6 (1968), both in France
Published works and other accomplishments
307 scientific journal articles, 23 book chapters, and advisory of 67 master’s dissertations and 50 PhD theses

Your career path has been wide-ranging. You have studied sea sponges, parasitic infection, and tissue regeneration. What challenges most interest you today?
I am at the end of my career and have resumed the line of research I started in Claude Lévi’s laboratory, at the University of Strasbourg, when I first immigrated to France. I am interested in cells. When I first arrived in Strasbourg, in the 1960s, Lévi was beginning to research the evolution of sea sponges, the first living beings to achieve multicellular organization. His goal was to understand how cell integration took place. Before sponges achieved this, cells functioned as isolated and autonomous entities. From here, they have become part of a system and perform only the role determined for them by the whole—or so they should.

What have you found?
Studying the organization of cell communities in sponges, I found they have a type of stem cell equivalent to the mesenchymal cells of more complex organisms. These cells do it all. They are able to multiply and become other types of cells. They reproduce and maintain their own population, but they also know what type of cell the organism needs at any given moment and generate it, supplying that need. In my PhD thesis, I proposed their existence in sponges. This type of cell is essential in cell therapy. The concept of a cell within its own context is the focus of my work because it applies to regenerative processes. To recover damaged tissue or restore aging-related damage, they must integrate with the surrounding tissue and perform a certain role. In regenerative medicine, this must be controlled and addressed. Today, my goal is to find out more about these cells and learn to manipulate them to promote regeneration.

How do the cells recognize what the organism needs?
The cells feel the effect of their environment, which gives them information. These cells often produce chemical mediators that spread and carry information over to other cells, influencing tissue conditions and generating systemic effects. More distant cells, in turn, produce mediators that are perceived by the initial cells. Based on the response they get from the tissue, these cells modulate their contact with the environment. This knowledge is critical. The cells to be used in therapy—introduced into a heart, for example—chemically perceive their environment. When it is diseased and has been damaged, the healthy cells introduced there interpret what needs to be done and proceed to repair the damage. The first clinical cell therapy I was a part of treated myocardial infarction.

Your work on cell therapy for the heart is widely cited. How did you become part of the project?
I joined the UFRJ Institute of Chemistry in 1980 to help cultivate cells for basic research on cell biology. They gave me a nearly fully set-up laboratory and I proceeded to ask colleagues from various institutions for cells to start the culture. I found that many were not well described or had unknown origins. So, I decided to establish the Rio de Janeiro Cell Bank—now the largest in Latin America, having categorized more than 300 cell lines. In the 1990s, a professor at the university, Halley Pacheco—a great Brazilian hematologist—implemented the bone marrow transplant service in Rio, which until then only existed at Hospital Israelita Albert Einstein, in São Paulo, and asked me to set up the cell preparation laboratory. These things brought me notoriety in the field. Later, Hans Dohmann, a cardiologist at Hospital Pró-Cardíaco, in Rio, and Emerson Perin, a Brazilian cardiologist at the Texas Heart Institute, in Houston, United States, raised the possibility of applying cell therapy to the myocardium. It was an interesting and original proposal. For legal reasons, they were unable to carry it out in the United States, so they came to Rio. They needed someone to manipulate the cells, and they asked me to do it.

The cell used in a therapy chemically perceives its environment and interprets what needs to be done.

Had you worked with the type of cells to be implanted in the heart before?
I had worked with this type of cell before in the bone marrow transplant service, at Hospital Clementino Fraga, at UFRJ. The difference is that the cells would be implanted in the heart instead of injected into the bloodstream. We performed the first experimental transplants for severe chronic heart disease, whose results were published in 2003. Both our group and that from Dusseldorf, Germany, were the first in the world to carry out myocardial cell therapy. At the time, we were not aware of each other, but we both achieved similar results. This work was very impactful. We then performed the first cell transplant for stroke and acute heart disease at Hospital Pró-Cardíaco.

This work gave rise to a 2004 national study by the Ministry of Health involving 1,200 participants, but little has been disclosed about the results. What happened?
The project was too ambitious. It escalated from 20 to hundreds of patients, covering four types of disease and including hospitals from Porto Alegre [Rio Grande do Sul] to Belém [Pará]. It was also too optimistic. The organizers did not assess the challenges faced by the project. From a scientific point of view, it was consistent, but hard to execute in a country as large as Brazil and with so many discrepancies in infrastructure. Multicenter studies are difficult to carry out even in other countries.

Did it achieve the results expected?
No. The myocardial infarction treatment confirmed previous results. INCOR [Heart Institute] participated in one branch of the cardiological study but failed to integrate well and left the project. The branch of the project that treated Chagas heart disease [caused by the parasite Trypanosoma cruzi], in Salvador, went ahead, but did not yield definitive results in the long run. The preparation of cells for transplantation is delicate and complex. Few hospital clinical laboratories can do it. At the time, I helped set up this type of service in the hospitals that were part of the study, with the exception of INCOR, but the proper handling of the cells cannot be taught in just a few days.

At that time, Brazil seemed to be at the forefront of regenerative medicine. How do things look today?
The initial phase was favorable. Brazilian legislation allowed for the introduction of a clinical trial of a new and original therapy, duly authorized by the national authorities and based on preclinical experimental manipulations. This permitted the Pró-Cardíaco project and the multicenter project, financed by the ministry, to proceed. We soon found—thanks to the multicenter study, in fact—that the quality of cell manipulation was critical to the results. We also found, both here and abroad, that cell manipulation procedures had to be regulated, both for preclinical trials and therapies. After these studies, several entities in China, Panama, and the Dominican Republic quickly began offering these therapies without any control. Following strict American standards, all of it was banned. Only laboratories that were certified—by ANVISA [Brazilian Health Regulatory Agency] in Brazil—and qualified as Cellular Technology Centers were allowed to manipulate these cells. The certification process was long and, at that time, it was hard to obtain approval for new clinical trials from CONEP [National Commission for Ethics in Research]. It progressively became a practice that was unauthorized for clinical use. Its use was potentially allowed in compassionate therapy, when there was no other satisfactory treatment, but even then it had to be done by a certified center, which did not exist. That was our low point.

Cell therapy works well for degenerative diseases that affect muscles, joints, and bones

Has this situation been resolved?
In our case, around 2006, Pró-Cardíaco set up the Excellion laboratory in Petrópolis. I came here to implement it, following the strictest international rules. It was the first laboratory for extensive cell manipulation for therapeutic purposes in the country to be certified by ANVISA. From that moment on, we were able to prepare cells for experimental protocols approved by CONEP. This allowed us to relaunch our therapies. ANVISA became effectively involved and, in 2008, published a resolution defining how such a laboratory should operate. This allowed the establishment of a network of qualified laboratories for cell manipulation. We had our work cut out for us. Nowadays, some of the laboratories work very well—like the one at the University of São Paulo [USP], in Ribeirão Preto, and the one coordinated by Paulo Roberto Brofman at the Pontifical Catholic University of Paraná (PUC-PR), which has become a benchmark for quality and provides cells for most clinical trials. Pró-Cardíaco was sold to Amil, later acquired by the United Health group, and Excellion was closed.

Has cell therapy reemerged in the country?
It has begun to; some types of therapy are becoming allowed. In 2015, the Federal Council of Dentistry authorized the use of fibrin- and platelet-rich plasma, which contains cells, to accelerate bone healing and formation. In recent years, ANVISA has published a set of resolutions defining the criteria for registering cell therapies that contemplate and define the extensive manipulation of the cells to be used in these treatments. The prospects are good, but it was found that even ANVISA-certified laboratories are not necessarily capable of handling cells properly due to a lack of experts. This is not an issue exclusive to Brazil, but we are about to take a leap and be able to offer this type of treatment—much like they are doing in Spain, South Korea, Australia, and Japan.

Are these therapies currently authorized for research use only?
For research, compassionate therapy, and clinical procedures with cell manipulation performed in properly certified institutions. I am part of a study group run by orthopedist Gildásio Daltro, from the Federal University of Bahia; they use stem cells to treat necrosis of the femoral head, a problem that usually affects young adults with sickle cell anemia and people of African descent, and usually requires the implantation of a metallic prosthesis. I have also worked with cell therapy for major burns at the Hospital da Força Aérea do Galeão. We were able to save the lives of many patients, including some who had burns on over 70% of their bodies. Professor Brofman’s group, in Curitiba, collaborates with hospitals that use cell therapy to treat diabetic ulcers. In cases where amputation is recommended, stem cells are extracted, manipulated, and reinjected into the patient. In up to half the cases, this has been enough to avoid amputation. In the United States, authorized therapies are still limited, while European countries, as well as Japan, South Korea, and Singapore, are beginning to offer cell therapies with the utmost rigor for specific cases and qualified patients. ANVISA is making a remarkable effort to authorize a series of advanced therapies in Brazil.

What disorders have been successfully treated with cell therapy?
It works well for muscle, joint, and bone issues, especially degenerative diseases. Generally speaking, the cells used are stem cells capable of generating bone and cartilage. There is a great demand for this type of treatment in sports medicine, as athletes often get injured. Clinical use in orthopedics is already approved in some countries, and I assume it will be one of the first authorized by ANVISA. Another area with potential for advances is the treatment of spinal cord injuries, which are uncommon but very visible. For hyperacute inflammation of the bowel, such as Crohn’s disease, it is possible to use allogeneic stem cells [from a donor] to reduce the inflammatory response. Pulmonary inflammation caused by COVID-19 also appears to respond well, as indicated by some experiments. Its use is also promising for acute myocardial infarction and stroke.

When working on integrating the university and industry, I realized that there was a lack of programs to train biotechnologists

You helped establish the biotechnology hub Bio-Rio. What was that like?
The idea for this biotechnology hub, which is located at the UFRJ Fundão campus, was to build a bridge between the basic science of the university and the private sector. I joined UFRJ right at the start of the discussion about working with the industry, which is absolutely necessary. A group of professors proposed building a technology park like the ones in foreign universities. I was part of the working group that put together the project and the administrative framework for Bio-Rio and later assessed the scientific value of the proposals. I quit after I joined the bone marrow transplantation service at the university hospital. I was later asked to join INMETRO, bridging the gap between the university and industry. In these roles, I realized that while we were discussing the relationship between the university and industry, a specific group of professionals was left out: biotechnologists. Brazil offered no higher education technical training for that profession. At the university, graduate students often fill this role, but industry requires technicians.

How was the issue resolved?
I contacted the Federal School of Education, Science and Technology, in Maracanã, and we created a training program for biotechnologists, which has now been around for 30 years. It was an interesting challenge. I sat down with Professor Maria Helena Nicola and we put together the project and curriculum and established the content for each subject—all in 10 days. The students get excellent training. Many have gone on to college later.

You were born in Zagreb when Croatia belonged to Yugoslavia. Do you recall anything from that time?
I was born on the outskirts of Zagreb, in November 1940. A week later, Yugoslavia was invaded by German forces and dragged into the war. My father was Serbian, and my mother was Croatian. Because he was Serbian and a military man, my father was seen by the Germans as someone to be executed. Zagreb was split into a poor side and a more developed side, which was forbidden to Jews, Romani, and Serbs. Our home was located on the rich side, but we could not be seen. I would spend the day out, far from home, with my grandmother, returning only at night. Sometimes, all we had to eat for a week—my parents, grandmother, sister, and myself—was a bag of corn. Despite these hardships, I had a happy childhood and adolescence. But I only returned there many years later. I immigrated to France in my third year of college and never went through mandatory military service in Yugoslavia. Until I was 36, I was considered a deserter, and risked being eliminated upon returning to Yugoslavia.

Your secondary education was focused on the humanities. What caused you to switch to biology?
In Zagreb, there was a traditional school of excellence, where the best students went. There, they were taught classical culture, Greek, and Latin—the latter was the official language of the Croatian government until the end of the 19th century. I was selected and attended that school. I had always had an interest in nature and was greatly supported in this by my paternal grandfather, an orthodox priest. After the war, I lived with him in Bosnia for a while. He had a farm and would take me for walks through fields and forests. When I began studying biology at the University of Zagreb, my goal was to work on the preservation of natural parks. Due to economic and social challenges, I decided to go to France, where I spent a short time as an illegal immigrant. In France, I heard about a scholarship for foreigners at the University of Strasbourg and applied for it. That was when I joined Professor Lévi’s group. Thanks to my final undergraduate papers, I was able to become a researcher at the CNRS [National Center for Scientific Research], which is heaven for any scientist. As I had become a French citizen, I had to complete military service. I already had a PhD and was much older than everyone else, so they assigned me to international scientific cooperation between France and Brazil.

In Zagreb, during the war, we sometimes had a single bag of corn to eat all week

So that is how you ended up here.
In February 1969, my life in Brazil began. I spent a short period in Recife at first, where there was some demand for my work; I also spent time on the Brazilian Navy research vessel Almirante Saldanha, studying marine biology. I was then sent to the Navy Research Institute, IPQM, on Ilha do Governador, in Rio. That was where I began working with Admiral Paulo Moreira, who was setting up a marine biology center in Arraial do Cabo, on the northern coast of Rio, in collaboration with France. IPQM had an agreement with the Walter Reed Army Institute of Research, in the United States, which at the time was developing a type of medicine that could be applied to the skin to protect soldiers from infection by the schistosomiasis parasite.

What was your role?
They asked me to cultivate cells to study worm penetration. That was how I began working with schistosomiasis. I loved Rio, so when I finished my military service, I remained there as a civil servant of the French Ministry of Foreign Affairs. At that time, there was another French-Brazilian project, set up by Admiral Aluízio Prata, to study schistosomiasis in a region where the disease was endemic, in Bahia. The French embassy transferred me to the Gonçalo Muniz Institute, which was privately owned [later incorporated into FIOCRUZ], where I began studying the pathology of schistosomiasis. We were guided by the Pasteur Institute of Lille, which was in the process of developing a vaccine. There, I had my first contact with the medical issues of poor populations in a tropical country. I was later asked by the World Health Organization [WHO] to join a tropical medicine committee to ensure integration between basic science and medicine. The immunology studies continued but, eight years later, the funding from Pasteur ran out and a vaccine was not developed. As a result, bilateral cooperation projects dwindled, and my work was reduced. In 1979, the civil cooperation service wanted to transfer me to another country.

Is it true that you were nearly sent to Iran?
At WHO meetings in Geneva, years before, I met biochemist Jacques Monod [1910–1976], from the Pasteur Institute, who had received the 1965 Nobel Prize in Medicine. He was on the committee with me. Monod asked me to come to Paris for a chat, where he asked me to set up the cell biology portion of the Pasteur Institute he was establishing in Tehran, under the patronage of Shah Reza Pahlevi [1919–1980]. Initially, leaving Bahia for Iran was unthinkable to me, but I ended up accepting. I then returned to Brazil because I had pending issues here. While deactivating my laboratory in Bahia, the Iranian Revolution took place, and Pasteur of Tehran was destroyed. The last plane for France that was able to leave Iran took the employees of the Institute, with nothing but the clothes on their backs.

Why did you decide to stay in Brazil?
I liked it here. The situation was complicated at Pasteur and my project in Bahia was no more. I went to Rio, joined CNRS for the second time and began organizing an international cooperation program involving UFRJ, where I started out as a visiting professor, and the Pasteur Institute in Lyon. I was incredibly happy at the Fundão campus.

What was the UFRJ like at that time?
Money was plentiful. We had great working conditions at the Chemistry Institute. I spent a few months in Rio and some in Lyon, but to be honest, I found it easier to work at Fundão. That was one of the reasons that led me to enter a public competition and stay at the university. At the time, we worked on long-term projects, which allowed us to sustain our research. Because I had no training in chemistry, I initially felt displaced at the institute, but the dialogue between the exact sciences and biomedical sciences became more and more relevant. I was responsible for the undergraduate courses on cell biochemistry, which also attracted biology and medical students, and for a long time I took over the coordination of the biochemistry graduate program there. These positions led me to work more and more closely with the medical field. I was very impressed with the quality of the work of the laboratories and was extremely optimistic about UFRJ. I had excellent relationships with groups from USP, the University of Campinas, and the Federal University of Rio Grande do Sul. I was asked by biochemist Ricardo Brentani [1937–2011], then director of the Ludwig Cancer Research Institute, to move to São Paulo, but I had a structure that worked very well at UFRJ. And, between you and me, I prefer Rio.

What is the university like today?
I am a professor emeritus. I do not work there much anymore, so I am not as aware of how things are. I had cancer in the early 2000s and retired. Then came my work at Excellion and INMETRO. Over time, I stopped frequenting the university, but I keep in touch. I think UFRJ needs to be reinvented. COPPE [Alberto Luiz Coimbra Institute for Graduate Studies and Engineering Research] works very well, but the health area is not so great. The university hospital appears to be in a difficult situation. We are going through a challenging time.

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