Directed differentiation of human-induced pluripotent (hiPSCs) into Leydig-like cells may be a novel and promising treatment for late-onset hypogonadism (LOH), according to a study recently published in Endocrinology.
Researchers led by Takashi Aoi, MD, Ph.D., of Kobe University in Japan, point out that LOH is characterized by various symptoms, including erectile dysfunction, decreased muscle mass and bone density, depression, and an overall poor quality of life. The condition has been treated with testosterone replacement therapy (TRT), but that treatment has its limitations. Injectable testosterone needs to be replenished every two to four weeks, the authors write, whereas ointment form of testosterone requires fewer hospital visits but everyday application, and the issue of secondary exposure of the coating formulation has been noted. Oral administration requires dosing 4 times in a day, and users must be alert for various adverse effects. “Furthermore, presently available TRTs do not restore the physiological fluctuation patterns of blood testosterone levels regulated by gonadotropin and gonadotropin-releasing hormone with feedback mechanisms,” they write. “Thus, novel therapeutic approaches as an alternative to TRT for LOH syndrome are required.”
Since testosterone is mainly secreted in the testes by Leydig cells, the researchers hypothesized that testosterone might be produced by artificially induced human Leydig cells, something the authors note that no one has tried or demonstrated before. “In the present study, we established a simple and robust differentiation protocol into functional testosterone-producing Leydig-like cells from hiPSCs via doxycycline-inducible overexpressing NR5A1 and showed not only marker gene expression but also the secretion of functional testosterone from the resultant cells,” they write.
For this study, the researchers used four iPSC clones: two male clones (3AB4, 73E1) and two female clones (201B7, 46C2-s4). They cultured the cells and added doxyclycine and a differentiation medium to the cells and performed marker gene expression and hormone secretion evaluations on the differentiated cells on day 21. “We succeeded in differentiating human iPSCs into Leydig-like cells with a simple protocol by expressing NR5A1,” the authors write. “Since our experimental system regulates the expression of NR5A1 in a doxycycline-dependent manner, NR5A1-iPSCs can be maintained and expanded and cryopreserved in the absence of doxycycline, and differentiation can be started promptly at any time by adding doxycycline.”
The researchers found that the differentiated Leydig-like cells were able to continue to secrete testosterone for four weeks after the end of differentiation. But there are still some issues to be resolved: namely, immune rejection and tumorigenicity. The testis is an immune-privileged site, so transplantation into the testis may overcome immune rejection, but there are still concerns about tumorigenesis. “Therefore, as is done for islet transplantation, encapsulating the iPSC-derived Leydig cells in a semipermeable device and then transplanting them into either the subcutaneous or peritoneal space may be a viable strategy for protecting cells from the recipient’s immune system and, moreover, for preventing invasion and metastasis in cases in which oncogenic transformation of the cells occurs,” the authors write.