Generation of Human Induced Pluripotent Stem Cells(iPS) using lentiviral vectors and Determination of Lentiviral Integration Profile

• Grant number: 09/13558-4
• Support Opportunities: Scholarships in Brazil - Master
• Start date: August 01, 2010
• End date: July 31, 2012
• Field of knowledge: Biological Sciences - Biology
• Principal Investigator: Elisa Maria de Sousa Russo
• Grantee: Luiza Cunha Junqueira Reis
• Host Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

Embryonic stem cells (ESCs), derived from the inner cell mass of blastocysts are pluripotent. They have great therapeutic potential as a source of tissues for transplantation and are an important tool for basic research to the study of cell differentiation and human development. The therapeutic effect of ESCs has been shown in animals, for different diseases. However, the clinical use of these cells in humans has not been reported. Issues such as immune compatibility between tissues derived from ESCs and from the receptor, remain to be resolved before they can be used in clinical trials. Recently, two groups reported the induction of pluripotency in human fibroblasts by transduction with viral vectors expressing different combinations of transcription factors (TF): Oct-4, Nanog, Sox2 and Lin28, or Oct-4, c-Myc, Klf4 and Sox2. The cells called induced pluripotent stem cells (iPS) have the morphology similar of the ESCs, express markers of pluripotency and are able to differentiate in vitro and in vivo in cells derived from three embryonic germ layers. The iPS are still inadequate for clinical use, but are an important tool for basic research. In this project, we intend to reprogram human fibroblasts by the addition of FT related to pluripotency. These factors can transform these cells differentiated / adult in pluripotent ones capable of unlimited expansion in vitro. We will use Lentiviral vectors and after the generation of iPS their morphological and functional characterization as well as the integration of lentivirus profile will be determinate using the technique of LM-PCR. The determination of sites of lentiviral integration is essential to assess whether there is influence of local integration in cell reprogramming. Less than 1% of cells that incorporate the virus become iPS. One possible explanation is that if iPS cells originate from progenitors or tissue stem cells that co-exist with the fibroblasts in culture. Another possibility is that besides the addition of transcription factors other factors are also needed to be activated by the lentiviral insertion. Candidates for such factors include Polycomb protein, which plays a critical role in maintaining pluripotency and factors involved in chromatin remodeling as ISWI and Brg1. The identification of these factors may provide that the production of iPS is more efficient.

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

REIS, Luiza Cunha Junqueira. Generation of human induced pluripotent stem (iPS) cell using lentiviral vector and determination of the lentiviral integration profile. 2012. Master's Dissertation - Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC) Ribeirão Preto.