Germ cells and the origins of mammalian pluripotent cells

Abstract

Mammalian embryonic stem (ES) cells originate from preimplantation embryos and can be propagated indefinitely without loss of pluripotency; i.e. the potential to develop into any embryonic cell type. ES cells have been described for mouse, rhesus monkey, and human. There is considerable interest in human ES cells because of their potential role in future medicine to regenerate damaged tissues. Potential risks need to be investigated in adequate model species, before such therapies can be practiced in human. The pig is a good candidate model organism in terms of organ sizes, physiology, and physiological life-span. However, true ES-cell lines have not yet been described for livestock species. It has been hypothesized that the resistance of livestock species to ES-cell derivation is caused by the difference in embryonic development between species. An important finding that is described in the thesis is that key transciption factors that are important for pluripotency in the mouse, are differentially expressed in early pig, cow, and, mouse embryo development. This could have consequences for the mechanisms of generation of ES-cells in livestock species. Spermatogonial stem cells (SSC) can be another source of pluripotent cells, if cultured under the appropriate conditions. The aim of one study of the thesis was to isolate, culture, and characterize male porcine germ cells. In particular, the effect of four commonly used growth factors on the self-renewal of porcine SSCs was investigated. In primary cultures of male germ cells, a large effect was found of growth factor FGF, which had a negative influence on the expression of germ cell specific genes and a positive influence on the expression of genes specific for somatic cells. Little is known about the mechanism behind the transition of SSC to ES-like cells, but the tumor suppressors TRP53 and PTEN seem to be involved in inhibiting this process. To get more insight in the role of these tumor suppressors in SSC, their expression levels were independently knocked down through RNA interference in a murine SSC line. In either case, this resulted in upregulation of the pluripotency factor Nanog. Therefore, it is hypothesized that male germ cells are insulated from pluripotency through independent suppression of Nanog through TRP53 and PTEN. In mouse development, expression of the pluripotency factor NANOG is restricted to cells of the pluripotent epiblast and to primordial germ cells, which are both cell types that can give rise to pluripotent cells. Although recent reports demonstrate that the testis can also give rise to pluripotent cells and testis function is closely associated with pluripotency, expression of NANOG in the testis has not been described yet. In a comparative study, NANOG expression was detected in differentiating male germ cells of mouse, dog, pig, and human. The findings of this study indicate at a conserved role for NANOG in the meiotic and post-meiotic phase of spermatogenesis. It is hypothesized that NANOG is involved in the epigenetic events that occur before and after meiosis.