Generation of canine pluripotent stem cells: factors involved in the establishment of reprogramming by gene induction

Author(s)

Natalia Juliana Nardelli Gonçalves

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina Veterinária e Zootecnia (FMVZ/SBD)
• Defense date: 2015-07-22

Examining board members

Carlos Eduardo Ambrosio; Fabiana Fernandes Bressan; Joaquim Mansano Garcia; Flavio Vieira Meirelles; Tathiane Maistro Malta Pereira

• Advisor: Carlos Eduardo Ambrosio

Abstract

The production of induced pluripotent stem cells (iPSC) from canine fetal fibroblast opens new ways for obtaining pluripotent cells and study its applicability for alternative therapies in veterinary medicine. In this context, this study investigated appropriate methods for producing pluripotent stem cells using a in vitro canine model (ESC-like), so far the production of true embryonic stem cells from ICM cultured blastocysts has not been fully characterized in domestic animals. The experiments aimed at increasing knowledge of the factors involved in reprogramming process in dogs, as well as the production of such strains and complete characterization. In the first experiment, a retroviral infection was compared to episomal reprogramming (never done for this specie) in an attempt to induce cells to pluripotency state without viral integration, also to observe the development of cells receiving separately the episomal plasmid plus transcription factors. The generation of colonies was possible only in the episomal plus c-MYC factor group, leading to increased cell proliferation producing iPS colonies with typical morphology and positive for the alkaline phosphatase detection. These results, so far as preliminary conclusions, are essential to obtaining strains without viral integration, increasing its applicability for clinical cell therapy. In the second experiment, we aimed to evaluate the OCT4 and SOX2 factors associated with fluorescent reporter proteins. These were analyzed by flow cytometry allowing the performance evaluation of each factor on the reprogramming process the fluorescence activated separation of cells containing the integrated gene, increasing the enriching the efficiency of reprogramming. Fluorescence microscopy analysis showed that the distribution of reporter protein was similar between the two different protein structures and not restricted to a particular cell region. OCT4 and SOX2 showed a high exogenous expression of each target gene, and double positive cells. However, no colony formation was observed at least 6 days after transduction. The last experimental chapter aimed to described the reprogramming mechanism of lentiviral integration to induce pluripotency in dog fetal fibroblasts. The lines obtained were fully characterized in this study, showing independency of LIF or any other supplemental inhibitors, resistance to enzymatic process (Tryple Express) and bFGF dependency only. A total of 66 clonal strains were obtained (hOSKM and h+mOSKM) while 10 (7 h+m and 3h) were maintained for 15 or more passages and used for in vitro characterization tests, with maximum efficiency of reprogramming 0.001% . All colonies were positive for the alkaline phosphatase detection, embryoid bodies formation, spontaneously differentiated and expressed high levels of endogenous OCT4 and SOX2. In vivo, the colonies were able to developed tumors 120 days after inoculation (confirmed via histopathology analysis), with predominantly mesodermal tissues. Chromosomal evaluations were made by FISH hybridization showing no chromosomal abnormality in iPSCs canine lines. The fully characterization of such lines as well as non-integrated experiments and factors associated via reporter proteins increases the knowledge of the iPSCs technology, establishing new strategies for more efficient and safe induction of pluripotency for potential use in cell therapy and clinical trials (AU)

• FAPESP's process: 11/22915-5 - Generation of canine pluripotent stem cell through in vivo and in vitro mechanisms
• Grantee: Natalia Juliana Nardelli Gonçalves
• Support Opportunities: Scholarships in Brazil - Doctorate