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Equine induced pluripotent stem cell (eiPSC) generation from episomal mechanism

Grant number: 19/03242-1
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): March 01, 2019
Effective date (End): March 31, 2020
Field of knowledge:Agronomical Sciences - Veterinary Medicine
Principal Investigator:Fabiana Fernandes Bressan
Grantee:Gabriela Barbosa
Home Institution: Faculdade de Zootecnia e Engenharia de Alimentos (FZEA). Universidade de São Paulo (USP). Pirassununga , SP, Brazil
Associated research grant:15/26818-5 - Investigation of cellular and molecular mechanisms on in vitro induced toti- and pluripotency acquisition - a translational approach, AP.JP

Abstract

Equines are animals of great economic and social importance for man, whether in activities related to production, sports, leisure, education and even therapeutic practice for the improvement of human health. Because they are directly related to physical effort, these animals are extremely susceptible to injuries and injuries in tendons, ligaments and bones and, as a consequence, can cause tissue fibroses resulting from the healing process, impairing the performance of these animals and contributing to recurrence of the lesions. To date the majority of treatments applied to this type of injury are not fully effective, so the search for new methods and treatment options are extremely necessary. In this context, induced stem cell (iPSC) represents a promising tool for both research and future applications in regenerative medicine. Initially, induction to pluripotency was based on an integrative system with viral vectors, however, this method can produce mutations that interfere in cellular function, influencing tumorigenicity. In view of this, researchers reported a noninclusive method of induction to pluripotency, applying the use of episomal vectors, where the cells generated are more safe for practical application, however, studies for equine cells are still scarce. Thus, the present study has as main objective to reprogram cells of adult equine fibroblasts through the system of induction to pluripotency with a nonintegrative method and to evaluate the maintenance of the pluripotency of these cells. IPSC will be generated from already cryopreserved fibroblasts in the Laboratory of Molecular Morphology and Development - LMMD, using an improved EBNA1/ OriP plasmidial system containing 5 sequences of reprogramming factors (Oct4, Sox2, Klf4, cMyc and Lin28). Cells will be transfected with plasmid vectors pEB-C5 and pEB-Tg by nucleoporation. After reprogramming, the cells will be morphologically characterized by alkaline phosphatase analysis by immunofluorescence and gene expression analysis will also be performed by qPCR techniques. At the end of this study, it is expected to establish and maintain reprogrammed equine fibroblast cells by a nonintegrative method. These results will represent the initial step towards the development of new methodologies to be applied in regenerative medicine.