Rapid Differentiation of Human Embryonic Stem Cells into Testosterone-Producing Leydig Cell-Like Cells In vitro

Background: Leydig cells (LCs) are testicular somatic cells that are the major producers of testosterone in males. Testosterone is essential for male physiology and reproduction. Reduced testosterone levels lead to hypogonadism and are associated with diverse pathologies, such as neuronal dysfunction, cardiovascular disease, and metabolic syndrome. LC transplantation is a promising therapy for hypogonadism; however, the number of LCs in the testis is very rare and they do not proliferate in vitro. Therefore, there is a need for an alternative source of LCs. Methods: To develop a safer, simple, and rapid strategy to generate human LC-like cells (LLCs) from stem cells, we first performed preliminary tests under different conditions for the induction of LLCs from human CD34/CD73 double positive-testis-derived stem cells (HTSCs). Based on the embryological sequence of events, we suggested a 3-step strategy for the differentiation of human ESCs into LLCs. We generated the mesendoderm in the first stage and intermediate mesoderm (IM) in the second stage and optimized the conditions for differentiation of IM into LLCs by comparing the secreted testosterone levels of each group. Results: HTSCs and human embryonic stem cells can be directly differentiated into LLCs by defined molecular compounds within a short period. Human ESC-derived LLCs can secrete testosterone and express steroidogenic markers. Conclusion: We developed a rapid and efficient protocol for the production of LLCs from stem cells using defined molecular compounds. These findings provide a new therapeutic cell source for male hypogonadism.