Brazilian study paves the way to new therapy for congenital anemia

Scientists prove that it is possible to transform the adult cells of carriers of the genetic mutation that causes the disease into pluripotent stem cells. The objective is to induce differentiation in blood stem cells for transplantation.

By Karina Toledo

Agência FAPESP – Aided by a Nobel Prize-winning technique for cellular reprogramming, Brazilian and American scientists managed to transform the skin cells of the carriers of a genetic mutation that causes aplastic anemia into induced pluripotent stem cells (iPSCs), similar to those found in embryos.

Also known as bone marrow aplasia, this potentially fatal disease is characterized by insufficient production of white blood cells, red blood cells and plaque. The symptoms are frequent infections, bleeding and severe anemia.

The objective is to find a way to transform iPSCs into hematopoietic stem cells (HPSCs) – which can become any type of blood cell – and infuse them into patients to promote bone marrow regeneration.

Currently, the sole therapeutic option in the case of congenital aplastic anemia is donor bone marrow transplantation, but only 25% of those affected by the disease find a compatible volunteer. In Brazil, an estimated 400 new cases of the disease appear annually.

The study was conducted by researchers at the Cellular Therapy Center (CTC) – one of FAPESP’s Research, Innovation and Dissemination Centers (CEPID) – at Universidade de São Paulo’s Ribeirão Preto Medical School (FMRP-USP) and included a partnership with scientists from the National Institutes of Health in the United States. The results were described in the most recent edition of The Journal of Clinical Investigation.

Shinya Yamanaka, of the University of Kyoto in Japan, described the cellular reprogramming technique in 2006. The method consists of inserting an adult cell into certain proteins that alter the expression of the cellular genome.

“These four transcription factors discovered by Yamanaka activate genes related to the embryonic stage of cells and shut off other genes that should be active after cellular maturation. However, we did not know if it would be possible to perform this reprogramming in carriers of the genetic mutation that causes aplastic anemia,” explained Rodrigo Calado, a CTC researcher who coordinated the investigation.

According to Calado, bone marrow aplasia can also be caused by autoimmune disease. In this case, the immune system destroys the bone marrow cells responsible for blood production, and the individual is treated with immunosuppressive medicine.

However, in the patients who participated in this study, the cause of the disease was a genetic defect responsible for the synthesis of an enzyme called telomerase, which is fundamental for maintaining the capacity of cellular proliferation.

“On the ends of chromosomes, there are structures called telomeres. They serve to protect the DNA, much like the plastic found on the tips of shoelaces. Every time the cell divides, the telomeres diminish in size until the cell can no longer proliferate and dies or enters into senescence (losing the capacity to divide). This is related to the process of aging,” explained Calado.

However, telomerase can maintain the length of telomeres even after cellular division. For this reason, it must be highly expressed in the embryonic phases and throughout the lives of stem cells that are in constant division. This is the case for bone marrow cells.

In patients with the genetic mutation, the hematopoietic cells suffer from a form of early aging and cannot proliferate adequately because there is not enough telomerase. Other parts of the body are also affected, and frequently, these people suffer from hepatic cirrhosis or pulmonary fibrosis.

“One of the objectives was to observe precisely what would happen with the telomeres during the process of cellular reprogramming. Theoretically, they should be longer because the cell undergoes a type of rejuvenation, or rather, it returns to the previous stage of its development,” explained Calado.

To verify this theory, the scientists reprogrammed the skin cells of two groups of patients: carriers of the genetic mutation with aplastic anemia and healthy volunteers who served as controls.

“We observed that in the control group, the telomeres doubled in size after cellular reprogramming. However, in the cells with the mutant gene, they continued on practically the same,” explained the researcher.

Another phenomenon observed by the group was that in reducing the level of oxygen in the incubators where pluripotent cells are stored, the size of the telomeres increased 20% in the two groups after one month.

“We reduced the concentration of oxygen found in air from 21% to 5%. This induced expression of a protein called HIF, which increased the synthesis of telomerase. Additionally, with less oxygen, DNA suffers less oxidation, and there is lower production of free radicals,” explained Calado.

Puzzle

Although the work has brought forward a series of promising and landmark results, many missing pieces of the puzzle remain to be discovered before this line of research will result in a therapy that can be tested in humans. One of the first obstacles to overcome is discovering a way to induce pluripotency in adult cells with the need to use a virus as a vector.

“In our study, as well as that of Yamanaka, we introduced into a virus the genes responsible for the expression of four proteins needed to reprogram the cell. The virus then integrated into the chromosome, and the cells began to synthesize these transcription factors. Some groups attempted to directly include the genes into the chromosomes of the cells, but the result was not that efficient,” explained Calado.

The part of the viral DNA responsible for causing the disease is removed before the procedure. Even so, it is a consensus among scientists that pluripotent cells obtained through this technique should not be tested in humans because of the risk of inducing tumor formation.

“These cells were tested in animals, and in some cases, tumors developed. The stem cells obtained in embryos were used in humans, and there were also cases of cancer,” explained Calado.

To minimize this risk, he affirmed, scientists must better investigate the mechanisms that regulate gene expression in stem cells, which is how we will gain greater control over gene behavior within an organism.

Another challenge, in the specific case of aplastic anemia, is finding a way to promote the transformation of iPSCs into HPSCs. “At the moment, we can only induce transformation in blood cells that are already differentiated, such as leukocytes, plaquettes and red blood cells,” commented Calado.

The article “Defective telomere elongation and hematopoiesis from telomerase-mutant aplastic anemia iPSCs” (doi:10.1172/JCI67146) can be read at www.jci.org/articles/view/67146.