Presentation Authors: Nicholas Deebel*, Guillermo Galdon, Nima Zarandi, Olivia Cornett, Winston-Salem, NC, Mark Pettenati, Winston-Salem , NC, Stuart Howards, Stanley Kogan, Anthony Atala, Hooman Sadri-Ardekani, Winston-Salem, NC
Introduction: Klinefelter Syndrome (KS) is typically defined as 47,XXY in the male patient. The onset of puberty in KS patients is associated with progressive testicular fibrosis, loss of spermatogonial stem cells (SSC), and impaired fertility. Previous work has demonstrated the ability to propagate in vitro SSCs in 2-dimensional (2D) cell culture. The objective of this study was to use propagated KS SSCs to form a functional 3D Human Testicular Organoid system (3D HTO) as a means of establishing a novel infertility treatment for KS patients.
Methods: In Vitro propagated KS testicular cells from 2D culture were thawed and recovered for one week. 3D HTOs were formed by placing 10,000 cells per HTO in ultra-low attachment plates for 48 hours. After the HTO formation period, they were placed in differentiating media for three weeks. Viability and structure of the HTOs were assessed with bright field and confocal microscopy as well as live/dead staining and ATP assays. Both qPCR and dPCR were used to confirm the presence and quantity of major testicular cells including spermatogonia (ZBTB16), Sertoli (SOX9), Leydig (TSPO) and peritubular (ACTA2)cells during all stages. In addition, HTO testosterone production was measured during culture. The presence of any haploid cell was assessed by post meiotic germ cells marker (PRM1) and X/Y/18 DNA FISH. The HTOs were assessed at day 0, 7, 14, and 21.
Results: Spherical HTOs were formed. Live/dead staining remained stable while ATP assays showed an initial decline due to transition from 2D to 3D system. PCR confirmed the presence of the four major testicular cells types. HTOS produced testosterone constantly. Furthermore, SSC differentiation was shown in each stage of the experiment by gene expression (ZBTB16 for undifferentiated, DAZL for differentiating, SYCP3 for meiotic and PRM1 for post meiotic germ cells). After 3 weeks, at least 12% of cells in HTOS were haploid (X/18 or Y/18).
Conclusions: This is the first study to demonstrate the ability to form stable and viable HTOs from human KS testicular cells. Furthermore, this system was able to functionally maintain four major testicular cell types and differentiate SSCs to post-meiotic cells. Future studies will focus on collecting viable haploid germ cells for use in intracytoplasmic sperm injection (ICSI).
Source of Funding: This work was supported in part by the Urology Care Foundation Research Scholar Award Program and AUA Southeastern Section.