Katja Eildermann

• Pluripotent stem cells are of outstanding interest for regenerative medicine due to their remarkable ability to generate all cell types of the body. Although pluripotent stem cells are classically derived from the inner cell mass of blastocysts, recent findings indicate that also spermatogonia can be a source for such cells. The initial aim of this project was to derive pluripotent stem cells from common marmoset monkey (Callithrix jacchus) spermatogonia. At the outset, the expression of the pluripotency factor SALL4 was investigated in marmoset embryonic stem cells and preimplantation embryos in mammalian germ line stem cells at different developmental stages and in adult mammalian spermatogonia. These experiments established SALL4 as an appropriate marker for pluripotent stem cells and undifferentiated premeiotic germ cells, including a subset of adult spermatogonia. Furthermore, these findings supported the hypothesis that SALL4-positive germ cells are a potential source of pluripotent stem cells. The experimental approach comprises the isolation and culture of spermatogonia including their eventual reprogramming and subsequent culture of spermatogonia-derived pluripotent stem cells. A prerequisite for this protocol is clear identification of the desired cells and their distinction from other cell types. Therefore, a detailed in situ analysis of marker expression in the adult primate testis was performed and an appropriate marker panel was established. All attempts to propagate spermatogonia or to derive pluripotent stem cells from testicular cells resulted in the enrichment of a fibroblast-like cell type. Initially these cells were thought to be pluripotent as two antibodies that detect the key pluripotency factor OCT-4 gave positive signals. Further analysis revealed that these signals were false-positive and a third antibody was required to display un-falsified staining. Using a standardized marker-panel and established differentiation assays, these cells were identified as testicular multipotent stromal cells (TMSCs). Importantly, TMSCs displayed the expression of many markers that are considered spermatogonia-specific within the testis, such as SSEA4, TRA-1-81, GFR-α, GPR125, THY-1 (CD90), and ITGA6. However, the germ cell-specific marker VASA and the spermatogonia-expressed factors MAGEA4, PLZF and SALL4 allowed clear distinction between marmoset spermatogonia and TMSCs. Furthermore, a culture-system for marmoset spermatogonia on irradiated TMSCs as a feeder layer was established. Although the propagation and reprogramming of marmoset spermatogonia to a pluripotent state was not achieved, this study provides for the first time a culture system for marmoset spermatogonia. Furthermore, a severe overlap in marker expression between spermatogonia and TMSCs was found. However, a panel of unequivocal markers for the identification of germ cells in culture was established, which provide a meaningful and reliable basis for future studies on cultured premeiotic (human and non-human primate) germ cells. Altogether, this work provides data which suggest that previous studies in the derivation of pluripotent stem cells from the human testes should be re-evaluated with regard to the suitability of the markers used for isolation and characterization of the cells.