Presentation Authors: HIMANSHU ARORA*, Evert Gonzalez, Joshua M Hare, RANJITH RAMASAMY, MIAMI, FL
Introduction: Impaired testosterone production as a result of Leydig cell loss or dysfunction can occur in men with testicular failure. Although several testosterone formulations are available, none are capable of replicating the physiological pattern of testosterone secretion. We have shown in our recent study conducted in murine models that, Leydig stem cell transplantation along with peritubular myoid cells and Sertoli cells could be used to physiologically increase serum testosterone thereby potentially minimizing the adverse effects. However, in order to optimize the function of Leydig stem cells, we need to understand the paracrine factors released by myoid and Sertoli cells. In the present study we evaluated the significance of paracrine factors secreted by human peritubular myoid cells and Sertoli cells on Leydig stem cell function.
Methods: Using an IRB approved protocol, about 10mg of testicular tissue from each of 5 men with testicular failure underwent testis biopsies for sperm retrieval were processed for Leydig stem cell isolation, culture and characterised. The presence of Leydig stem cells (LSCs), Sertoli cells (SCs) and peritubular myoid cells (PMCs) in the harvested cellular pool was validated by immunofluorescence and quantitative real time PCR (qPCR). CD146 (+) cells representing LSCs were sorted using MACS kit and maintained along with unsorted cells. Condition media was collected from both the cell types and screened for secreted protein using Human Antibody Array.
Results: We successfully isolated and cultured LSCs from all 5 testis biopsies. We were able to culture up to 3 million cells / biopsy. Of the cells cultured, up to 70% of the cells were Leydig stem cells and 10% of them were Sertoli-cell in origin on day 14. IF and qPCR data showed as the majority of cell population was undifferentiated (PDGFR-Î±). Upon stimulation by LH, the expression of 3Î²HSD (mature Leydig cells) was increased and that of PDGFR-Î± was decreased. Importantly, human antibody protein array demonstrated increased expression of specific cytokines in the media of LSCâ€™s that were co-cultured with Sertoli cells and myoid cells compared to the media from purified LSCs culutres (CD146 positive).
Conclusions: Our results indicate that LSCs can be isolated and cultured from men with testicular failure. There are specific paracrine factors which are released by adjacent Sertoli and myoid cells which could be critical for LSC differentiation and testosterone production. Further studies are ongoing to validate the implications of these paracine factors in terms of their role in LSCs function, differentiation and survival.
Source of Funding: American Urological Association Research Scholar Award and Stanley Glaser Award to RR. J.M.H. is supported by NIH grants 1R01 HL137355, 1R01 HL107110, 1R01 HL134558, 5R01 CA136387, 5UM1 HL113460 and Soffer Family Foundation.