Analysis of the germ cell potential, in vitro and in vivo, of somatic derived stem cells

Current understanding of germ cell development is hindered by several factors including: a limited number of germ cells present in developing organisms, difficulties in isolating and culturing germ cells, and the complex migration and formation of germ cells, in vivo. Models for in vitro germ cell formation may provide insights into understanding germ cell biology and development.;In order to determine if the ability of somatic stem cells to form germ cells is a cross species and developmental stage phenomena stem cells were isolated from newborn mouse skin. These cells are morphologically similar to the fetal porcine skin-derived stem cells and express similar markers. Under very different in vitro culture conditions the mouse isolated stem cells were able to form OLCs that express markers consistent with early stage oocytes.;The ability of newborn mouse skin-derived stem cells to contribute to the germline in vivo was also investigated. Oct4-GIP skin derived stem cells were used as donor cells to test their ability of chimeric contribution to the germline, as well as organ development. Following injection into wildtype blastocysts and subsequent transfer to pseudopregnant recipient mice, pups were born. Analysis of the founder pups obtained confirmed the contribution of donor cells to various tissues including the gonads in some mice. In order to confirm germline contribution the founder pups were bred to wildtype mice. The resulting f1 mice produced were analysed and some found to contain donor cell contribution in their genomes. This confirmed the ability of newborn skin-derived stem cells to contribute to the germline in vivo.;The overall objective of my Ph.D. research program was to study if stem cells isolated from skin have the intrinsic machinery to differentiate into oocyte-like cells. Porcine fetal skin derived stem cells were first used as a model. When induced to differentiate, a subpopulation of these cells expressed pluripotent markers associated with germ cell formation. During differentiation large cells with a morphology characteristic of oocytes ware detected as well as follicle-like structures that secreted estradiol, progesterone, and responded to gonadotropin stimulation. Further characterization of these oocyte-like cells (OLCs) revealed similarities and differences with regards to their expression of germ cell markers. During late stage differentiation, blastocyst-like structures were detected. While this suggests the ability of a small subpopulation of OLCs to complete meiosis, further investigation revealed that the majority of OLCs failed to reach late stages of meiosis and lacked the expression of several meiosis markers. This suggests that the OLCs produced in vitro are not perfect oocytes and require additional factors to complete meiosis.