The effect of endocannabinoids on the differentiation and proliferation of spermatogonial stem cells and their precursor primordial germ cells from human induced pluripotent stem cells

Male factor is responsible for 50% of infertility cases, while 15% are due to germ cell aplasia resulting in no sperm. Assisted reproductive techniques (ART) protocols are inadequate for men with germ cell aplasia to have children. Although human pluripotent stem cell (hiPSC)- derived primordial germ cells (hPGCs) and spermatogonial stem cells (hSSCs) provide an autologous germ cell source for infertile men, however, their yield in vitro is low. The endocannabinoid system (ECS) is present in mouse embryonic stem cells and human testicular microenvironment, but the potential distribution and involvement of ECS in hiPSC, hiPSC- derived hPGCs and hSSCs remain unknown. In this study, we hypothesized that ECS involvement could expand the germ cell pool and stimulate the proliferation and maintenance of the hiPSCs, hiPSC- derived hPGCs, and hSSCs. To test this hypothesis, the human dermal fibroblasts (hDFs) were reprogrammed into hiPSCs carrying markers of pluripotency and Blimp1+, VASA+ hPGCs, ID4+, PLZF+ hSSCs were differentiated from hiPSCs and characterized by qPCR, flow cytometry (FCM) and immunofluorescent (IF) labeling. The hiPSCs, hPGCs, and hSSCs secrete 2-AG and AEA at picomolar to nanomolar levels by liquid chromatography/mass spectrometry. The qPCR, FCM, and IF revealed the expression of CB1R, CB2R, TRPV1, and GPR55 receptors of hDFs, hIPSCs, hPGCs, and hSSCs at various levels. CB2R expression is higher in hSSCs compared to all other receptors and hDFs, hiPSCs, hPGCs. CB2R agonists increase hiPSCs proliferation in line with the receptor distribution pattern compared to other receptor agonists. The EC50 of CB65 was determined as 2.092x10-8 M on 78 hours by real-time analysis. CB65 maintains the pluripotency of hiPSCs and stimulates the differentiation of ID4+ hSSCs, PLZF+ SSPCs, and SCP3+ spermatocytes on days 10 and 12 when applied at submaximal dose. This study demonstrated for the first time that CB2R agonism increases the yield of hiPSCs and hiPSC- derived hSSCs. Enrichment of the male germ/stem cell pool with CB65 may be a part of ART protocols in clinic for infertile men having complete germ cell aplasia.