FGFR3 as a marker for human spermatogonial stem cells, basic research for human application

• Applicant: Dr. Kathrein von Kopylow
• Subject Area: Reproductive Medicine, Urology
• Term: from 2014 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 264290960

The true identity of human spermatogonial stem cells (hSSCs) could not be uncovered to date. On the other hand, therapeutical application of this cell type may be useful. By microarray analysis (MA), we identified the Fibroblast growth factor receptor 3 (FGFR3) as a human spermatogonial specific marker. FGFR3 is expressed on the cell surface of A type spermatogonia (SPG). Immunofluorescent multiple staining followed by confocal microscopy demonstrated the appearance of the protein only within non-proliferating and non-differentiating SPG and the coexpression with the pluripotency marker UTF1. Additionally, FGFR3 protein is only expressed in small spermatogonial clusters, as revealed by whole mount preparations of seminiferous tubules. Hence, a potential stem cell state of the FGFR3+ human SPG is indicated. We developed and optimized a method to obtain a pure FGFR3+ hSSC population without somatic contaminations from small testis biopsies by combining magnetic cell isolation and subsequent picking of bead-labeled cells using a micromanipulator. To show stem cell characteristics of the bead-labeled cells, these were immuno-stained for the pluripotency factor UTF1 and resulting in a clear positive nuclear staining in contrast to the UTF1-negative, bead-unlabeled cells within the negative fraction. In addition to this, live/dead staining demonstrated the viability of the isolated potential hSSCs. Furthermore, using single-cell PCR we could verify the bead-labeled SPG as FGFR3-expressing also on mRNA level compared to the bead-unlabeled cells without FGFR3-expression. Aside from this, FGFR3+ isolated cells could be cultivated by maintaining their cellular morphology for 70 days and their UTF1 pluripotency marker expression for 51 days maximum (different experiments). Using MA data, Pearson correlation analysis to the FGFR3 expression uncovered the Fibroblast growth factor 9 (FGF9) as a potential ligand of FGFR3 in human SPG. Within our above mentioned cell culture experiments FGF9 was already successful tested. FGF9 could promote self-renewal division of hSSCs.In this project, we want to optimize the culture conditions for the FGFR3+ SPG to propagate the potential hSSCs in their undifferentiated state. Therefore, we want to utilize established protocols for hSSC proliferation beside own approaches with growth factors in different combinations and concentrations. Cellular expansion of the potential hSSCs should serve as a prerequisite for xenotransplantation which is the only approach to reveal hSSC characteristics. Analysis of the epigenetic state and the marker expression of pre- and post-cultivated cells should identify and therefore exclude any cellular changes during cell culture. The new findings are anticipated to gain knowledge about the mechanism of human spermatogenesis and may have implications for a therapeutical approach with hSSCs.

• DFG Programme: Research Grants
• Cooperation Partner: Professor Ulrich Zechner, Ph.D.