Modelling the developing gonad –organoid- and iPSC-based approaches

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Human pluripotent stem cell-derived primordial germ cell-like cells (hPGCLCs) currently represent the best available model to mimic early human germline developmental processes in vitro and allow experimental setups to deeper understand germ cell specification and underlying molecular pathways. The male fate in primordial germ cells is induced during testicular morphogenesis. Sertoli cells engulf germ cells and subsequently form aggregates as the first sign of seminiferous cord formation. Germ cells in seminiferous cords respond with a male-specific pattern of expansion and migration and implement transcriptional and epigenetic changes. Here, we aim to describe the role of candidate genes identified within the CRU in early PGC specification. Using protocols established during the first funding period, we will generate human iPSC lines with a loss of function (LoF) mutation in identified candidate genes via the CRISPR/Cas9 system, and analyze morphological features, gene expression, and interaction with somatic cells to identify pathways associated with impaired fertility. As second objective, we will extend our xeno-organoid approach, established within the first funding period. The reconstituting rat seminiferous tubule-like structures mimic testicular morphogenesis in vitro and allow co-culture of wildtype or mutated PGCLCs within a testicular microenvironment. We thereby expect to describe crucial factors and genes involved in male germ cell differentiation. As endpoints for characterization of male fate in PGCLCs, we apply imaging and histological endpoints using markers to describe male-directed differentiation, migration, and clonal expansion of PGCLCs. Additionally, changes in transcription and methylation will be determined before and after exposure to the ex vivo male microenvironment. Third, we will extend and diversify the generation and characterization of the somatic testicular microenvironments. To extend the observation period, the rat organoids will be subjected to xenografting into immunodeficient mice. Additionally, we will apply human primary testicular cells from adult transgender patients to generate seminiferous tubule-like structures in vitro and thus establish a standardized homologous culture system. Our studies will generate a new approach and new findings to better understand human germ cell development. With a focus on sex determining mechanisms, we foresee to describe pathways potentially affected in male infertility. The study provides novel insights into basic cellular and molecular mechanisms controlling gonadal sex-differentiation and interplay of germ cells with somatic tissues. The data have therefore immediate and direct clinical relevance since incorrect differentiation of germ cells may lead to partial or complete germ cell loss and infertility.