Manipulation of inhibitory pathways of pluripotency induction aiming efficiency increase in iPSs generation process

• Grant number: 10/02616-0
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: July 01, 2010
• End date: November 30, 2014
• Field of knowledge: Biological Sciences - Genetics - Human and Medical Genetics
• Principal Investigator: Marco Antonio Zago
• Grantee: Mariane Serra Fraguas
• Host Institution: Hemocentro de Ribeirão Preto. Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da USP (HCMRP). Secretaria da Saúde (São Paulo - Estado). Ribeirão Preto , SP, Brazil
• Associated scholarship(s): 13/19545-7 - MiR-29a target two central components of active DNA demethylation, TET1 and TDG: potential mechanism during reprogramming, BE.EP.DD

Abstract

Embrionary Stem Cells (ESC) are capable of self-renewal keeping the pluripotent state. These properties make ESC a potential tool for Regenerative Medicine. Recently, the generation of induced pluripotent stem cells (iPSs) by somatic cell genetic reprogramming (trough Oct4, Sox2, c-Myc and Klf4 genes), created new expectations for cellular therapy. However, beside the genetic risks associated with retroviral vectors integration in the human genome, this process occurs with an extremely low efficiency, hindering its routine application. It is known that some pathways characteristic of pluripotent stem cells (like WNT/Gsk3-²/²-Catenin) can interfer in the process of iPSs induction. Thus, the inhibition (or activation) of some os theses pathways in somatic cells submited to the process of pluripotency induction could result in an increased induction efficiency. The RNAi (interference RNA) tecnique is a tool whose specificity allows the direct evaluation of diferents components of a given pathway. Thus, this project aims to evaluate the influence of the WNT/Gsk3-²/²-Catenin pathway in the efficiency of iPSs generation. For this, interference RNA agaisnt Gsk3-² and ²-Catenin, to respectively, activate and inhibit the pathway, on fibroblasts submited to the process of pluripotency induction using lentiviral vectors. The efficiency obtained in the induction of these fibroblasts, determined based on the number of colonies with ESC-like morphologie, will be compared to that obtained using fibroblasts transfected with inespecific RNAi, to non transfected cells, and also to cells treated with a Gsk3-² inhibitor. iPSs colonies will be characterized trough their genetic expression by Real Time PCR. With these results we expect to better understand the process of pluripotency induction, and also, to evaluate the use of RNAi in process. Finally, the increase of efficiency in this process can lead to the expansion of its use in basic and clinical research. (AU)

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)

FRAGUAS, MARIANE SERRA; EGGENSCHWILER, RETO; HOEPFNER, JEANNINE; DOS SANTOS SCHIAVINATO, JOSIANE LILIAN; HADDAD, RODRIGO; BOURGUIGNON OLIVEIRA, LUCILA HABIB; ARAUJO, AMELIA GOES; ZAGO, MARCO ANTONIO; PANEPUCCI, RODRIGO ALEXANDRE; CANTZ, TOBIAS. MicroRNA-29 impairs the early phase of reprogramming process by targeting active DNA demethylation enzymes and Wnt signaling. STEM CELL RESEARCH, v. 19, p. 21-30, MAR 2017. (10/02616-0, 13/19545-7)