Germ cell development in vitro (WP2)

In analogy to recently published mouse studies, this work-package aims to establish in vitro spermatogenesis from human testicular biopsies in an organoid or 3D cell culture system and investigate the differentiation from spermatogonia to sperm and the balance between SSC self-renewal and differentiation as well as the influence of somatic cells. Furthermore, the aim is to gain more insights into the process of in vitro differentiation of germ cells from ESCs and iPS cells.

In vitro derivation of PGCs from ESCs and iPS as a model system allows the generation of sufficient cell numbers to perform robust biochemical analysis of protein-protein and protein-mRNA interactions during further differentiation of PGCs to more advanced germ cells. We will explore the mechanism of the developmental switch from mitosis to meiosis, which is one of the most challenging steps to induce in sperm development. We will characterize cell-cell interactions of testicular cells in cell and organoid culture in combination with xenografting. Molecular mechanisms and factors involved in cord formation and interaction between different cell types, especially between somatic and germ cells, will be characterized to get more insight on the control of germ cell development.

Additionally, we aim to identify biomarkers during the course of differentiation, using next-generation sequencing followed by in-depth bioinformatics analysis. In addition, computational tools that allow a system-biological approach will be employed to identify meaningful pathways and networks, as well as interactions and interconnecting nodes that may serve as either drug targets for differentiation induction and/or platforms on which reporter assays can be developed. Potential drug targets will be validated using knock-down approaches in reporter assays to systematically identify compounds that enhance the differentiation potential of germ cells in developing and optimizing the various culture systems (from iPS, germ cells or organ culture).