HIMANSHU ARORA, Marilia Sanches Santos Rizzo Zutti, Bruno Nahar, Joshua M. Hare, Ranjith Ramasamy
INTRODUCTION AND OBJECTIVES
Impaired testosterone production as a result of Leydig cell loss or dysfunction can occur in men with testicular failure. Testis failure is typically seen in men with Klinefelter syndrome and in men undergoing high dose chemotherapy or hematopoietic stem cell transplant. Currently, these patients are offered long-term testosterone supplementation that can cause infertility. We evaluated an approach for isolation and differentiation of Leydig stem cells from men with infertility that underwent testis biopsies.
A total of 6 men with testicular failure underwent testis biopsies for sperm retrieval. Using an IRB approved protocol, about 10mg of testicular tissue from each of these men were processed for Leydig stem cell isolation and culture. Leydig stem cells and Sertoli cells were analyzed by immunofluorescence (IF) and quantitative real time PCR (qPCR) for PDGFR-a and Sox-9 respectively. After stimulation by Luteinizing hormone (LH), we compared the levels of 3ßHSD mRNAs (involved in testosterone production) using qPCR, and testosterone production in the media using radioimmunoassay from the adult Leydig cells.
We successfully isolated and cultured Leydig stem cells from all 6 men with testicular failure who underwent testis biopsies. Leydig stem cells were maintained in the media without LH for up to 30 days. We conducted a minimum of five independent isolations within 30 days. We were able to culture up to 3 million cells / biopsy in 14 days. 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, the expression of PDGFR-a was high. Upon stimulation by LH, the expression of 3ßHSD was induced and that of PDGFR-a was reduced at both RNA as well as at protein levels.
Our results indicate that Leydig stem cells can be isolated and cultured from men with testicular failure. Leydig stem cells can be differentiated with LH and the adult Leydig cells can be functional. These results suggest that Leydig stem cell therapy can be used to increase serum testosterone without affecting fertility outcomes