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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-\u03b2 signaling pathway is a critical regulator of testis development that can be harnessed to regulate the testicular cells\u2019 behavior. Based on our previous finding on the crucial role of TGF-\u03b2 inhibition in promoting spermatogonia proliferation and differentiation, we have developed a novel approach to improve TO development from dissociated testicular cells.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Methods<\/jats:title>\n            <jats:p>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-\u03b2 inhibitors, SB431542 (SB) or LY2157299 (LY).<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>According to our results, TGF-\u03b2 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-\u03b2 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-\u03b2 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.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusion<\/jats:title>\n            <jats:p>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.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/s13287-025-04513-0","type":"journal-article","created":{"date-parts":[[2025,8,19]],"date-time":"2025-08-19T22:54:05Z","timestamp":1755644045000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Harnessing TGF-\u03b2 signaling to improve testicular organoid development from dissociated testicular cells"],"prefix":"10.1186","volume":"16","author":[{"given":"Seyedeh-Faezeh","family":"Moraveji","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Saiedeh","family":"Erfanian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohammad Hossein","family":"Ghanian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6528-3687","authenticated-orcid":false,"given":"Hossein","family":"Baharvand","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,8,20]]},"reference":[{"issue":"5","key":"4513_CR1","doi-asserted-by":"publisher","first-page":"942","DOI":"10.1093\/biolre\/ioab014","volume":"104","author":"TC Cham","year":"2021","unstructured":"Cham TC, Chen X, Honaramooz A. 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