New ramifications

Brazilians dominate technique to transform adult stem cells into embryonic cells

Celula_Gustav_Klimt_032'Tree of life’, Gustav Klimt, 1905-1909/detail from a sketch for the wall frieze at the Stoclet Palace in Brussels Every year the prestigious journal, Science, elects the major accomplishments of the scientific year. In the list published in December 2008 the highlight was cell reprogramming, a technique that returns embryonic characteristics into adult cells. As a result of this transformation a skin cell, for example, becomes capable of creating any other body tissue, a property that until now was restricted to cells removed from embryos in the first days of their development. The reprogrammed cells are called induced pluripotent stem cells (iPS). The innovative technique, which ideally will reduce the need for extracting cells from human embryos, was developed in 2007 by Japanese scientist, Shinya Yamanaka, from the University of Kyoto. Less than two years later, at the end of January, two Brazilian groups announced that they had also been successful in this area. One of them is neuroscientist, Stevens Rehen, from the Federal University of Rio de Janeiro (UFRJ), in collaboration with bio-doctor, Martin Bonamino, from the National Cancer Institute (Inca). The other group, the research of which is coordinated by Dr. Dimas Tadeu Covas and funded by FAPESP, is from the Hemocenter of the University of São Paulo (USP) in Ribeirão Preto. There, biologist Virginia Picanço, doing post-PhD studies at the Cell Therapy Center (CTC), one of the Research, Innovation and Disclosure Centers (Cepids), the funding for which comes from FAPESP, got the cells in November and is working to reproduce the same achievement and increase the stock. In doing so Brazil is getting closer to the group of countries where researchers are already using this technique.

In Rio de Janeiro, Rehen and Bonamino, with the help of post-graduate students Bruna Paulsen, from UFRJ, and Leonardo Chicaybam, from Inca, have reprogrammed renal cells from  a strain that already forms part of the  basic equipment of many laboratories worldwide. They celebrated their success in obtaining cell groupings that have the three embryonic germ layers, the endoderm, mesoderm and ectoderm, from which all tissues in the organism originate. It was the sign they had obtained pluripotent cells. The group also reprogrammed skin cells (fibroblasts) from mice, an important model in medical research. Before they had even completed the tests for obtaining specialist cells and testing their functioning the group chose to publicize their results to the Brazilian scientific community. The announcement was made discreetly on January 22 on the website of the National Embryonic Stem Cell Laboratory (Lance), which is linked to the Ministry of Health. They posted their recipe at so that other research groups could produce their own strains of iPS. Two days later the news spread with a report that was published by the Estado de S.Paulo newspaper.

The researchers from Rio, who received support from the Research Protection Foundation of the State of Rio de Janeiro (Faperj), Inca and the National Council for Scientific and Technological Development (CNPq), used a virus as a Trojan horse to reprogram renal cells in order to insert into them four DNA fragments capable of activating genes that normally function in embryonic cells. That’s where Bonamino’s team came in; it specializes in producing vectors capable of causing genetic changes in cells. The group from Inca uses cells known as packaging cells, as tailor-made virus factories: in addition to carrying an extra gene, these viruses do not have the part of the genetic material that allows them to reproduce and attack the cells. “The virus we built is like a car that carries the gene to the cell. There, it is disassembled but as it doesn’t carry any assembly instructions it is unable to replicate itself”, explains Bonamino.

Virginia Picanço, from the CTC, used the same technique but inserted six genes in the cells, instead of four. She herself built the viruses, inoculated human fibroblasts and waited for changes in the cells to appear. After 11 days, with the help of geneticist Lygia da Veiga Pereira, from USP, she confirmed that the fibroblasts had started functioning genetically as embryonic cells.

One of the problems with this method is that the viruses may insert genes in any region in the DNA, often compromising the action of genes that are fundamental to the cells as tumor suppressors. Previous research had already shown that animals generated from reprogrammed cells have 20% more chances of developing tumors. Another essential aspect is the number of viruses that can infect each cell; it has to be sufficient so that some copies of each of the genes are inserted into each cell. Even using a substance that accelerates the reprogramming (valproic acid) Bruna Paulsen says that the 30,000 human cells used in the research only produced 10 iPS colonies. The process was also less efficient in mice: 250,000 cells generated 48 colonies of induced cells.

“We’d liked to have shown that we managed do the bread and butter work in an effective manner”, says Rehen. Before using these cells in clinical research, however, it will be necessary to check that they can generate normal specialist cells. Furthermore, the protocols used in Rio de Janeiro and Ribeirão Preto are only likely to function for fibroblasts and renal cells. It will probably be necessary to adjust the recipe to manipulate other types of cell. Cardiologist José Eduardo Krieger, from USP’s Heart Institute (InCor), is trying to apply this “bread and butter” approach to human fat cells and other strains he works with, but he has had no results as yet.

To grow
Bonamino is planning to use other types of viral vector that can be controlled, by using a mechanism, for example, that allows researchers to link and unlink genes. “We’re looking for more efficient, easier and safer ways of manipulating cells”, is how he sums it up. He’s not alone. Biologists Eugenia Constanzi-Strauss, from the Institute of Biomedical Sciences at USP, and Bryan Strauss, from InCor, in a partnership with Lygia Pereira and Mayana Zatz, from USP, intend inserting the same four genes used in Rio into a type of virus that transports genes without integrating its own genome to that of the manipulated cell. These are adenoviruses, which are already used in the United States for this purpose.

Rehen also intends comparing the performance of the iPS with embryonic stem cells to see if they really function in the same way. “Research with embryonic cells is very much more advanced. That’s undoubtedly where the first important results are going to appear and the first promising therapies”, he forecasts. For Dr Dimas Tadeu Covas, director of the Hemocenter in Ribeirão Preto, this should not be an argument for not studying alternatives. He says that it’s necessary to investigate embryonic, adult and reprogrammed stem cells in parallel. “Reprogramming is all the rage at the moment, but we don”t know yet what type of cell is most efficient for each line of research”, he comments.

Even though the accomplishment represents an important technical advance everybody agrees that the most interesting is yet to come: using the induced cells to answer scientific questions. The idea is that they can be used for studying diseases and testing drugs in test-tubes. “We’re not going to inject them into sick people”, Rehen emphasizes. Each researcher will be able to use them to build research models in his or her specific area. The group from UFRJ, for example, intends taking advantage of this additional tool for studying Parkinson’s disease and testing possible therapies against it. According to Bonamino, various groups from Inca will be able to use the technique to produce different tissues from the cells of patients with a family predisposition to cancer, to understand how the disease manifests itself in different parts of the organism and seeks ways of fighting it. At InCor, Krieger intends producing models for studying little known cardiac diseases and testing the drugs that are effective for each patient. In Ribeirão Preto the objective is to study the formation of blood cells.

“The scientific community is very happy to get hold of these cells. The promise is that all will have access to them”, celebrates geneticist Mayana Zatz. She wants to use this system to help understand and treat neuromuscular diseases. “I can compare the functioning of cells from two patients that have the same mutation and different clinical situations”, is the example she gives. At the Human Genome Studies Center she has a cell bank, with adult stem cells and fibroblast from more than 200 patients. Perhaps this collection will now be worth even more.

Over and above the prospect of obtaining reprogrammed cells what Mayana values is scientific collaboration. In an area in which each technique and each stage demand a great level of specialization, isolated laboratories have fewer chances of producing high impact results. This is why everybody comes out a winner with the initiative for making the recipe for producing iPS readily available. “But this will only be effective if the government maintains the funding”, warns the researcher, who is worried about recent threats of sizeable cuts in federal funding for scientific research.

The first clinical study in humans
Therapy based on embryonic stem cells will be tested on patients in the USA

Marcos Pivetta

In an historic decision a company in California, the Geron Corporation has received authorization from the Food and Drug Administration (FDA), the body that regulates drug use and the sale of food in the United States, to test a therapy based on human embryonic stem cells on people. The company is going to inject these cells into ten people who are paralyzed because of serious damage to the spinal cord and analyze the safety and possible effects of the procedure. This is the first time that a therapy with this type of cell has received the green-light for use in humans.

“The decision marks the beginning of what is potentially a new chapter in medical therapy that goes beyond pills and reaches a new level of cure: the restoration of the function of an organ or tissue by injecting healthy substitute cells”, said Thomas B. Okarma, president of Geron, in a press communiqué on January 23.

The company intends injecting the cells in the location of the lesion in the bone marrow in people who have lost movement from the thorax down. The therapy will be administered to those taking part in the clinical study between 7 and 14 days after the lesion occurred. There’s evidence that the chance of a favorable result is greater in patients who have recently suffered an accident. Researchers at this stage have no illusion of returning all movement to patients with this possible therapy, since its effectiveness has not yet been proved.

Embryonic stem cells can be transformed into any type of body cell and tissue. But it’s not known what reactions they may cause when introduced into the organism, nor if it will be possible to control their transformation into more specific cells. There’s a risk they may cause tumors. Furthermore, some religious people and even scientists question whether it is ethical to use discarded human embryos for obtaining these cells that seem to have enormous therapeutic potential.

As the FDA has given the go-ahead for the first clinical trials in humans it is understood that the possible benefits of this new therapy outweigh the risks and moral issues. Approval of the study just three days after the swearing in of the new president of the United States, Barack Obama, a defender of scientific and technological research, was interpreted as a coincidence by executives from Geron.  But there were those who thought that the green light for the study reflects the position of the new incumbent of the White House. Since 2001, restrictions against embryonic stem cell research that were adopted by George W Bush have been in force in the United States.