Presentation Authors: Nicholas Deebel*, Guillermo Galdon, Nima Zarandi, Winston-Salem, NC, Mark Pettenati, Winston-Salem , NC, Stuart Howards, Winston-Salem, NC, William Kearns, Baltimore , MD, Stanley Kogan, Winston-Salem, NC, Christina Wang, Yanhe Lue, Ronald Swerdloff, Torrence , CA, Anthony Atala, Hooman Sadri-Ardekani, Winston-Salem, NC
Introduction: Klinefelter Syndrome (KS) is defined by aneuploidy (most frequently 47,XXY) in the male patient. The onset of puberty in KS patients is associated with testicular fibrosis, loss of spermatogonial stem cells (SSC), and impaired fertility. While it is feasible to cryopreserve SSCs from KS patients for future fertility treatments such as SSC transplantation or in vitro spermatogenesis, adaptation of current SSC propagation systems for KS patients must first be demonstrated. This work demonstrates the in vitro culture of mouse and human KS SSCs.
Methods: KS postnatal mouse testicular tissue was obtained from the existing UCLA model. KS human tissue was obtained from the experimental testicular tissue bank at Wake Forest Baptist Health. Selected patients underwent peripheral blood karyotyping and were demonstrated to be non-mosaic 47,XXY. Testicular cells were extracted from cryopreserved tissue and propagated in long-standing culture based on our previous work with normal human testes. These cells were further characterized with q-PCR, digital-PCR, Flow Cytometry and Magnetic Activated Cell Sorting, next generation sequencing (NGS)-based molecular karyotyping and X/Y chromosome FISH staining.
Results: Both mouse and human KS cells were individually isolated and propagated in culture for at least 80 days. The presence of Spermatogonia, Leydig, Sertoli, and Peritubular cells were confirmed with cell specific gene expression. The population of ZBTB16+ undifferentiated spermatogonia was identified throughout all stages of culture. This was reinforced by the detection of HLA-/CD9+/CD49f+ SSCs throughout culture. FISH staining for X/Y chromosomes showed that most cells in culture were XXY. However, subpopulations of XY and XX cells were observed; representing 4% and 2% of all propagated cells respectively. The XX and XY subpopulations were enriched with MACS for CD9 as an enriched spermatogonia marker. NGS testing showed all cells being 47,XXY (with 90% sensitivity for mosaicism).
Conclusions: To the best of our knowledge, this is the first study to demonstrate the successful isolation and propagation of mouse and human KS testicular cells, including SSCs. It is our belief that this will expand the therapeutic arsenal for azoospermia in KS patients through both in vitro or in vivo avenues.
Source of Funding: This work was supported in part by the Urology Care Foundation Research Scholar Award Program and AUA Southeastern Section.