Bovine pluripotent stem cells: strategies for generation and differentiation towards germ cells

The use of pluripotent stem cells for bovine transgenesis as well as the induction of primordial germ cell (PGC) differentiation from pluripotent bovine cells, represents conceptually feasible alternatives to reach high economic impact goals such as generating transgenic animals with improved or economically valuable traits or the modification of the genetic pool in an isolated population through germ cells transplantation. The goal of this study was to contribute towards the understanding of nuclear reprogramming and the germ cell specification in vitro in the specific case of Bos Taurus species (cattle) species. In this manuscript, key concepts on pluripotency, reprogramming and germ cell differentiation are introduced and reviewed (Chapter I). Focused mainly on the current murine and human models and what is known about cattle ; followed by a description on the materials and methods used in a regularly bases during the project (Chapter II). Then, the generation and characterization of three male biPSC cell lines by an integrative approach (lentiviral) with the potential to differentiate to several issues in vitro and in vivo is described followed by attempts to generated bovine induced pluripotent stem cells (biPSC) using a non-integrative (episomal) approach iPSC. Finally, different culture conditions and culture media were evaluated in terms of their effect on key pluripotent genes expression by the biPSC produced (Chapter III). The results demonstrate it is possible to increase the expression level of REX1, OCT4, NANOG and SOX2; key markers of pluripotency by pharmacological inhibition of the Glycogen synthase kinase-3 (GSK-3), the extracellular signal-regulated kinase (ERK) / mitogen-activated protein kinase (MAPK) pathways and DNA methyltransferases (DNMTs). The same effect is observed for the expression of OCT4 and REX1 merely by culture under LIF conditions. Next, a system for biPSC germ cell differentiation and isolation, based on VASA gene expression, was developed (Chapter IV). In this part of the work, an in silico analysis and in vitro validation of the putative regulatory elements upstream of the bovine vasa homolog gene (Bvh) was performed. This was through the modelling of regulatory elements by bioinformatics tools. Using this approach, a regulatory region of approximate 0.4 kb upstream of Bvh was identified. This region encloses a core of TFBS conserved, at the functional level, between at least three mammalian species. These findings were validated in human seminoma cells in vitro and subsequently the validated candidate regulatory sequences were used to generate male biPSCTg(bvh-EGFP) cell lines (germ cell reporter cell lines). The germ cell potential, across the cell lines generated, was evaluated (Chapter V) under distinct culture conditions (Hanging drop and suspension) and germ cell differentiation inducers such as Retinoic Acid (RA) and BMP4. The second part of the chapter involved analysis of the gene expression profile of the sorted population undergoing PGC specification. Using the culture conditions evaluated it was concluded that the biPSC generated not only had germ cell potential but also their rate of differentiation towards PGCs increases ten times in the presence of either RA or BMP4. Furthermore, those cells undergoing germ cell differentiation resemble an early post migratory state of PGC. The final chapter (Chapter VI) is a summary of the main findings from the previous three chapters. It centres on the potential of biPSC as a source of germ cells in vitro and the identity of the cells undergoing differentiation. Its identity is elucidated in the light of what is known from human and murine models. Finally, the limitations of this work and the future avenues of research are discussed