Harnessing TGF-β signaling to improve testicular organoid development from dissociated testicular cells.

BACKGROUND: Testicular organoids (TOs) have generated great interest in reproductive biology as a reliable experimental tissue model for pharmaco-toxicology studies and therapeutic applications. However, current TOs mostly fail to recapitulate the native testicular architecture and function, likely due to an imbalance between the spermatogonia and functional niche cells. TGF-β signaling pathway is a critical regulator of testis development that can be harnessed to regulate the testicular cells' behavior. Based on our previous finding on the crucial role of TGF-β inhibition in promoting spermatogonia proliferation and differentiation, we have developed a novel approach to improve TO development from dissociated testicular cells. METHODS: The testicular cells were isolated from prepubertal mice, encapsulated within inner core of Matrigel-based core-shell hydrogel droplets hanging from filter inserts, and exposed to the small molecule TGF-β inhibitors, SB431542 (SB) or LY2157299 (LY). RESULTS: According to our results, TGF-β inhibition considerably improved formation of spherical-tubular structures (STSs), resembling the compartmentalized architecture of native testicular tissue, as indicated by increased number, size and average area of the STSs. The TGF-β inhibitor-derived TOs (TiTOs) revealed more profoundly a tissue-specific spatial expression pattern of testicular markers and superior steroidogenic activity in response to gonadotropin stimulation (3.69-fold and 3.00-fold vs. the untreated control in the SB and LY-treated groups, respectively). The stimulatory effects of the TGF-β inhibition were attributed to the promoted proliferation of cells, as demonstrated by up-regulation of cell cycle promoting genes, down-regulation of proliferation inhibitor genes, and up-regulation of proliferation genes, as well as increased number of proliferative germ cells in the treated TOs. CONCLUSION: This work presents a simple and efficient method for development of well-organized and functional TOs which can be investigated as a complementary treatment with any other TO culture systems.