Brazilian and U.S. researchers team up to develop a safe line of stem cells to treat marrow lesions

Results from a USP and North Carolina State University study could help treat genetic diseases, such as muscular dystrophy.

By Karina Toledo

Agência FAPESP – Obtaining safe cell lines and pluripotent stem cells for clinical use, for example, in the treatment of medullary lesions and genetic diseases such as muscular dystrophy, is the main objective of a project that is bringing together researchers from Universidade de São Paulo (USP) and North Carolina State University.

Professor Carlos Eduardo Ambrósio of the USP’s Zootechny and Food Engineering School in Pirassununga presented preliminary results during FAPESP Week North Carolina on November 12, 2013.

“We are using cells from adult animals to induce pluripotency. Our focus is to obtain a safe line, without tumor-formation potential, for use in preclinical and clinical trials,” explained Ambrósio.

The project, coordinated by Ambrósio and Jorge Piedrahyta of the North Carolina State University School of Veterinary Medicine, was one of those approved in the most recent call for proposals launched under the auspices of the University Global Partnership Network (UGPN). This international consortium, formed by USP (Brazil), North Carolina State University (U.S.) and the University of Surrey (U.K.), aims to support research collaboration among the three institutions.

The project is also linked to a FAPESP Regular Research Grant coordinated by Ambrósio in Brazil.

To induce pluripotency in the fibroblasts of dogs, the researchers used the Nobel-prize-winning technique (2012) that was described in 2006 by Shinya Yamanaka of Kyoto University in Japan. The method consists of inserting certain proteins capable of reprogramming the cellular genome – known as transcription factors – into a cell obtained from adult skin.

These transcription factors activate genes related to the embryonic stages of the cell and deactivate other genes that are activated after cellular maturation.

The adaptation of the technique to obtain canine induced pluripotent stem (iPS) cells was described in an article published in the journal Reproduction in Domestic Animals. The study generated another article in the journal Placenta.

“The technique described by Yamanaka proposes the use of four different transcription factors: OCT3/4, SOX2, KLF4 and C-MYC. However, at the same time that this method induces pluripotency in the cell, it also makes the cell teratogenic [capable of inducing teratomas; tumors comprising mixed tissues]. What we are now attempting in in vitro experiments is to obtain an iPS cell using a reduced number of transcription factors. We believe that this approach can reduce the carcinogenic potential of the cell. We also intend to generate numerous Brazilian and American lines and to conduct studies on the reactions of the cultured cells to different growth factors, as well as to analyze the gene sequences of these cells,” explained Ambrósio.

Another of the group’s strategies is modulating the cellular genome using tools such as transcription activator-like effector nucleases (TALENs), a group of enzymes that make it possible to activate or deactivate certain genes of interest. This study is being conducted in North Carolina as part of the doctoral research of FAPESP fellow Natalia Juliana Nardelli Gonçalves, under Ambrósio’s mentorship. Gonçalves conducted the first stage of the study during a FAPESP research fellowship abroad.

During the master’s studies of FAPESP fellow Helena Debiazi Zomer, who was advised by Ambrósio, the project focused on obtaining iPS cells from rabbit stem cells in adipose tissue.

Stem cells

Between 2008 and 2011, Ambrósio conducted research under a Young Investigator Research Grant, examining several methods to obtain mesenchymal stem cells – cells capable of differentiating into different types of tissue, such as bone, cartilage and fat – from several species of animals.

The study resulted in the creation of a stem cell bank that comprises more than 10 lines of cells from dogs, cats, sheep and pigs. This archive is now connected to the Molecular Morphophysiology Laboratory and Development FZEA, coordinated by Professor Flávio Vieira Meirelles, and the new Clinical Hospital Didactic Unite, conceived with the intention of generating therapeutic innovations for veterinary uses.

“In addition to cellular therapy, we also tested gene therapy using the tools of gene editing in cultures of cells from dogs with muscular dystrophy. We tried to correct the defective gene that causes the disease in vitro,” explained Ambrósio.

In Ambrósio’s opinion, the generation of knowledge regarding the manipulation of stem cells and gene editing could contribute to the treatment of chronic and genetic diseases in animals, with future applications in humans.