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However, its sexual reproduction, culminating in infectious oocysts, occurs exclusively in feline intestines. Recent studies identified the transcription factors AP2XII-1 and AP2XI-2 as crucial for pre-sexual development. Their depletion enabled merozoite formation in human fibroblasts, but progression to sexual stages appeared to require additional cues. Host-specific factors governing this process are suspected but remain elusive. Here, we describe a robust continuous feline intestinal organoid culture system without feeder cells to investigate whether the feline cellular and metabolic environment promotes sexual development of in vitro-generated merozoites. Using ultrastructural and transcriptional analyses, we found elevated levels of sexual stage-specific transcripts. While advanced sexual stage formation could not yet be observed, our feline intestinal organoid model provides a controlled and reproducible experimental system to systematically uncover the feline host factors and molecular mechanisms of\n                    <jats:italic>T. gondii<\/jats:italic>\n                    \u2019s\u00a0sexual development.\n                  <\/jats:p>","DOI":"10.1038\/s42003-026-09710-y","type":"journal-article","created":{"date-parts":[[2026,2,14]],"date-time":"2026-02-14T21:45:13Z","timestamp":1771105513000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Long-term feeder cell-free cat intestinal organoid cultures to study Toxoplasma gondii\u2019s sexual development"],"prefix":"10.1038","volume":"9","author":[{"given":"David","family":"Warschkau","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2858-4776","authenticated-orcid":false,"given":"Tobias","family":"Hoffmann","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6474-9139","authenticated-orcid":false,"given":"Michael","family":"Laue","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Antonia","family":"M\u00fcller","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chandra","family":"Ramakrishnan","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Giulia","family":"Rigamonti","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fabrizia","family":"Veronesi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elvio","family":"Lepri","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mohamed Ali","family":"Hakimi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Christian","family":"Klotz","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2271-3699","authenticated-orcid":false,"given":"Frank","family":"Seeber","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2026,2,14]]},"reference":[{"key":"9710_CR1","doi-asserted-by":"publisher","first-page":"109","DOI":"10.1126\/science.364.6436.109","volume":"364","author":"M Wadman","year":"2019","unstructured":"Wadman, M. Closure of U.S. Toxoplasma lab draws ire. Science 364, 109 (2019).","journal-title":"Science"},{"key":"9710_CR2","doi-asserted-by":"publisher","first-page":"e0173018","DOI":"10.1371\/journal.pone.0173018","volume":"12","author":"PS Guiton","year":"2017","unstructured":"Guiton, P. S., Sagawa, J. M., Fritz, H. M. & Boothroyd, J. C. An in vitro model of intestinal infection reveals a developmentally regulated transcriptome of Toxoplasma sporozoites and a NF-kappaB-like signature in infected host cells. PLoS ONE 12, e0173018 (2017).","journal-title":"PLoS ONE"},{"key":"9710_CR3","unstructured":"de Muno, M. R. et al. Development of schizont stages of Toxoplasma gondii in primary cell culture of feline enterocytes. in Towards New Perspectives on Toxoplasma gondii (eds El-Ashram, S. Tellez-Isaias, G. Alali, F. & Alouf, A.) 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AP2XII-1 is a negative regulator of merogony and presexual commitment in Toxoplasma gondii. mBio 14, e0178523 (2023).","journal-title":"mBio"},{"key":"9710_CR7","doi-asserted-by":"publisher","DOI":"10.1128\/msphere.00606-22","volume":"8","author":"S Srivastava","year":"2023","unstructured":"Srivastava, S., Holmes, M. J., White, M. W. & Sullivan, W. J. Jr. Toxoplasma gondii AP2XII-2 contributes to transcriptional repression for sexual commitment. mSphere 8, e0060622 (2023).","journal-title":"mSphere"},{"key":"9710_CR8","doi-asserted-by":"publisher","unstructured":"Jiang, Y., Shi, Y., Xue, Y., Hu, D. & Song, X. AP2XII-1 and AP2XI-2 suppress schizogony gene expression in Toxoplasma gondii. Int. J. Mol. Sci. 25, https:\/\/doi.org\/10.3390\/ijms25105527 (2024).","DOI":"10.3390\/ijms25105527"},{"key":"9710_CR9","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-44967-z","volume":"15","author":"JL Wang","year":"2024","unstructured":"Wang, J. L. et al. 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