Research on stem cell biology

The Center for Research in Cell Therapy focuses on advanced and applied research on stem cell biology. Processes will be developed for the large-scale production under good manufacturing practice conditions.

By Claudia Izique

Agência FAPESP - In early 2000, the survival rate of patients suffering from acute promyelocytic leukemia (APL) in Brazil was 50% compared to the greater than 80% survival rate among cases reported in the United States and in European countries.

However, higher incidence of the disease in Latin American countries did not justify the discrepancy in the statistics: APL responds well to treatment with all-trans retinoic acid (ATRA), a medication that is distributed through the National Healthcare System (SUS).

The explanation for the discrepancy between the outcomes was due to late diagnosis and the resulting delay in beginning treatment for a disease that leads to severe hemorrhagic syndrome and an increased number of deaths.

By combining basic and clinical research, researchers at the Center for Research in Cell Therapy (CTC), a Research, Innovation and Dissemination Center (RIDC) funded by FAPESP and housed at the School of Medicine at the University of São Paulo (USP) in Ribeirão Preto, proposed a new diagnostic model adopted by a consortium composed of eight public hospitals in five Brazilian states.

“In nine years, mortality decreased by half, and patient survival increased to 70%,” researcher Eduardo Magalhães Rego says with satisfaction. The results of the study made the cover of the March 14, 2013 issue of the journal Blood. The protocol has become standard in other Latin American countries, and the consortium also includes hospitals in Chile, Uruguay and Mexico and over the next few months will include Paraguay and Peru.

The new APL diagnostic is the embodiment of one of the key missions of the Center for Research in Cell Therapy and the RIDC Program, established by FAPESP in 2000: develop excellence in research with a commitment to the applied results.

“The CTC has established a fertile environment in which to develop methodologies related to research into cancer, stem cells, biotechnology and molecular biology,” notes Marco Antonio Zago, the Center’s coordinator and dean of research at USP. “We conduct basic research and have a clinic for treating patients and laboratories at the Hemocenter and at Hospital das Clínicas, in addition to a bone marrow transplant unit.”

The formulation of the new APL diagnostic test entailed the journey from the laboratory to the clinic. “APL is the result of breaks in two chromosomes, with the pieces being exchanged with each other, forming a fused gene. The challenge was in understanding how this gene causes leukemia,” Rego explains.

Using transgenic models, researchers were able to reproduce the disease in mice and determined that some proteins that should act as suppressors were not functioning. One of them, the promyelocytic leukemia (PML) protein, which is “imprisoned” in a well-defined structure in healthy individuals, appears to be dispersed in victims of the disease and does not regulate the body.

In fact, PML dispersion was key to the diagnostic test, which is conducted by examining the reaction of bone marrow antibodies “donated by an Italian researcher,” Rego continues. “It is an immunofluorescence laboratory test that is available in even medium-sized hospitals and produces results within six hours,” Rego states.

The next step will be to test the effect of hematopoietic stem cell transplants (HSCTs) extracted from the bone marrow to treat opportunistic infections in carriers of acute myeloid leukemia.

Cell differentiation

The CTC has worked on developing HSCT therapy since early 2000. “At the time, no one talked about stem cells. We just used marrow transplants,” Zago recalls. “These came from three sources, marrow cells, blood cells and placenta cells, and each of them responded differently in the cell treatment.”

The team has focused its study on cell differentiation and clinical response. In 2004, when a Swedish study suggested that stem cells could reduce the body’s immune response because they altered T lymphocytes, the team decided to assess the use of stem cells in the treatment of diabetes, an autoimmune disease in which the immune system attacks the insulin-producing cells of the pancreas. The hypothesis was that if the attack was stopped and the remaining cells preserved, it would be possible to use HSCTs to restore the pancreas, reduce the dependency on insulin and avoid the common complications of the disease, such as retinopathy, nephropathy and neuropathy.

The first experimental protocol involved 25 patients diagnosed with Type 1 diabetes. Prior to undergoing chemotherapy, hematopoietic stem cells were collected from their bone marrow and frozen to reset the immune system and stop the assault on the pancreas.

The frozen stem cells were then transfused, producing new marrow and new blood cells. Of the 25 patients, three no longer receive insulin; 22 went back to using insulin after a certain period of time but at a lower dose than that used prior to the treatment.

A new HSCT protocol using a new chemotherapy cocktail was initiated in 2009 with four other patients whose Type 1 diabetes had been diagnosed less than five months before. The study is being conducted in partnership with Chicago’s Northwestern University and Paris’ Diderot University. “We are excited about the results,” says researcher Maria Carolina de Oliveira Rodrigues.

In 2008, in another experimental protocol, the researchers attempted to modulate the immune system of patients with diabetes using mesenchymal cells, which are present in all organ tissues and are extracted from the bone marrow of a relative.

“The hypothesis was that mesenchymal cells would be able to migrate and differentiate into the pancreatic cells that produce insulin,” Rodrigues told Agência FAPESP. The response, however, was less than satisfactory, and a new study may be conducted.

A protocol for the National Healthcare System

A protocol that is similar to that for Type 1 diabetes has also shown to be promising in the treatment of multiple sclerosis, a disease that attacks the central nervous system and progressively compromises neurological capacity. Since 2002, 100 patients have received chemotherapy and the intravenous injection of hematopoietic stem cells to stop the advance of the disease, explains researcher Rodrigues.

Over the last 11 years – the period that corresponded to the FAPESP funding of the CTC – administration of this chemotherapy was being calibrated according to the patient’s response. “A very toxic therapy was replaced by a more suitable one. We also learned that the transplant was not successful when the patient was at a very advanced stage.” The current protocol is being evaluated jointly with Northwestern University, and the results are being compared to those of the best drugs.

The CTC has also achieved excellent results in the treatment of systemic scleroderma, which progressively affects the cells of the connective tissue, causing vascular alterations and fibrosis of the skin. Although the conventional treatment with cyclophosphamide hinders the progression of the disease, in at least one third of the cases, HSCT preceded by chemotherapy is the recommended procedure. In 48 patients, the transplant stopped the assault and reversed the associated condition of cutaneous degeneration, thus stabilizing the patient. “This protocol is on its way to being adopted by the SUS,” says Rodrigues.

However, the risks involved must be weighed. In February 2013, in partnership with researchers from Northwestern University, the CTC published an article in the journal Lancet that recommended conducting a thorough cardiac evaluation to better assess the appropriateness of transplants as a result of the chemotherapy cocktail, among other factors. “We will begin a second study to compare three chemotherapy schemes and determine which is best,” states Rodrigues.

The prominence and good results achieved by the study should be attributed to the highly trained team, such strong international partnerships as those with the Universities of Montreal, Guelph, Munchen, King's College, Leiden and, in particular, the long-term large-scale project funding.

“The team is competitive. During this period, in addition to support from FAPESP, we also received financing from other research-sponsoring agencies, such as the National Council for Scientific and Technological Development (CNPq) and the Brazilian Innovation Agency (FINEP), not to mention the University itself. The outcome is that the funds allocated by FAPESP have doubled,” Zago acknowledges.

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* This is the first in a series of special reports about FAPESP's RIDC Program regarding the Centers supported from 2001 to 2013.