Differentiation of human umbilical cord mesenchymal stem cells into Leydig-like cells with defined molecular compounds

95% of the body’s testosterone is produced by the Leydig Cells (LCs) in adult testis, and LC functional degradation can cause testosterone deficiency ultimately leading towards hypogonadism. The transplantation of LCs derived from stem cells is a very promising therapy to overcome the testosterone deficiency. The isolated umbilical cord mesenchymal stem cells (UMSCs) were identified by flow cytometry and adipogenic and osteogenic differentiation. Western blotting and reverse transcription polymerase chain reaction (RT-PCR) were used for the differentiated Leydig-like cell identification. The comparisons of the testosterone levels, gene expression levels, and cyclic adenosine monophosphate (cAMP) productions were performed through radioimmunoassay, quantitative polymerase chain reaction (qPCR), and cAMP assay kit, respectively. Here, it is stated that our isolated human UMSCs, which could positively express CD29, CD44, CD59, CD90, CD105, and CD166 but negatively express CD34 as well as could be differentiated into adipocytes and osteocytes, could be differentiated into Leydig-like cells (UMSC-LCs) using a novel differentiation method based on molecular compounds. The enrichment UMSC-LCs could secrete testosterone into the medium supernatant and produce considerable cAMP at the stimulation of luteinizing hormone (LH), and positively expressed LC lineage-typical markers LHCGR, SCARB1, SATR, CYP11A1, CYP17A1, HSD3B1, HSD17B3, and SF-1 as well as negatively expressed mesenchymal stem cell typical markers CD29, CD44, and CD105. The expression levels of NR3C4, PDGFRA, and NR3A1 in UMSC-LCs were higher than those of UMSCs and were comparable with LCs. These results illuminated that UMSCs could be differentiated into Leydig-like cells using the defined molecular compounds, which might further support MSC-derived Leydig cell transplantation therapy for testosterone insufficiency.