Abstract
Male hypogonadism arises from the inadequate production of testosterone (T) by the testes, primarily due to Leydig cell (LC) dysfunction. Small molecules possess several advantages, including high cell permeability, ease of synthesis, standardization, and low effective concentration. Recent investigations have illuminated the potential of small molecule combinations to facilitate direct lineage reprogramming, removing the need for transgenes by modulating cellular signaling pathways and epigenetic modifications. In this study, we have identified a specific cocktail of small molecules, comprising forskolin, DAPT, purmorphamine, 8-Br-cAMP, 20α-hydroxycholesterol, and SAG, capable of promoting the conversion of fibroblasts into Leydig-like cells (LLCs). These LLCs expressed key genes involved in testosterone synthesis, such as Star, Cyp11a1, and Hsd3b1, and exhibited the ability to secrete testosterone in vitro. Furthermore, they successfully restored serum testosterone levels in testosterone-castrated mice in vivo. The small molecule cocktails also induced alterations in the epigenetic marks, specifically H3K4me3, and enhanced chromosomal accessibility on core steroidogenesis genes. This study presents a reliable methodology for generating Leydig-like seed cells that holds promise as a novel therapeutic approach for hypogonadism.