Effects of low oxygen tension in the reprogramming efficiency and manutention of induced pluripotent stem cells in equine

• Grant number: 18/04009-6
• Support type: Scholarships in Brazil - Doctorate
• Effective date (Start): June 01, 2018
• Effective date (End): June 30, 2020
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Reproduction
• Principal researcher: Joaquim Mansano Garcia
• Grantee: Raquel Vasconcelos Guimarães de Castro
• Home Institution: Faculdade de Ciências Agrárias e Veterinárias (FCAV). Universidade Estadual Paulista (UNESP). Campus de Jaboticabal. Jaboticabal , 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

As being a species currently involved with physical work, equines are frequently affected by tendon, ligament, and bones injuries and other diseases as osteoarthritis, conditions still without effective treatment options, and that can even lead to a loss of motor function. Thus, studies related to regenerative medicine are significant for this species. Induced pluripotent stem cell (iPSC) technology can be used as a promising tool both for basic research and regenerative medicine. However, it is not an always successful procedure, being that some cells could be in an intermediate state between the pluripotency and the differentiated cell. In view of the importance of studies of methods that complete and help the cellular reprogramming, since it brings basic knowledge that lead to better efficacy in iPSC cell production, and that the oxygen tension employed in cell culture and reprogramming has influence in the development and maintenance of iPSC, adipose tissue cells from adult equines are going to be reprogrammed through two methods, one employing lentiviral vectors (VL) with the 4 transcription factors [Oct4, Sox2, Klf4 e c-Myc (iPSC 4FT)] and the other employing episomal vectors, which is a method free of integration to the cell genome. Intending to apply iPSC in regenerative medicine, the use of nonintegrative system matters. Currently, there is no report in the literature of equine iPSC cells if not by integrative vectors, nor the influence of oxygen in reprogramming or culture of this cells, which makes the present study essential. Both iPSC reprogrammed by the VL and episomal method are going to be divided in cultures in conditions: 5% CO2, 5% O2 and 90%N2, or 5%CO2, 20%O2 and 75%N2. The obtained colonies will be characterized by the efficiency of the reprogramming process, differentiation capacity in vitro and in vivo, immunophenotyping, and evaluation of gene expression. In addition, to evaluate the influence of the oxygen, the activation of the glycolytic pathway, the presence of free radicals, mitochondria morphology, the presence of mitochondrial membrane potential, global methylation of DNA and of microRNAs profile are going to be evaluated, as well as a karyotyping to investigate possible chromosomic alterations. By the end of the evaluations, it will be possible to demonstrate the alterations caused by the oxygen and clarify its influence in the reprogramming process and maintenance of the equine iPSC.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

DE CASTRO, RAQUEL V. G.; PIERI, NAIRA C. G.; FANTINATO NETO, PAULO; GRIZENDI, BIANCA M.; DORIA, RENATA G. S.; MEIRELLES, FLAVIO V.; SMITH, LAWRENCE C.; GARCIA, JOAQUIM M.; BRESSAN, FABIANA F. In Vitro Induction of Pluripotency from Equine Fibroblasts in 20% or 5% Oxygen. STEM CELLS INTERNATIONAL, v. 2020, NOV 26 2020. Web of Science Citations: 0.