{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T17:11:04Z","timestamp":1790788264480,"version":"4.1.0"},"reference-count":21,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2022,4,5]],"date-time":"2022-04-05T00:00:00Z","timestamp":1649116800000},"content-version":"vor","delay-in-days":4,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2022,4]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The generation of T cells is a complex, carefully orchestrated process that occurs in the thymus. The ability to mimic T cell differentiation <jats:italic>in vitro<\/jats:italic> has opened up avenues to better understand different stages of thymopoiesis but has also enabled the <jats:italic>in vitro<\/jats:italic> production of mature T cells suitable for immunotherapy. Among existing protocols, the artificial thymic organoid (ATO) system has been shown to be the most efficient at producing mature conventional T cells. In this serum\u2010free model, human or murine hematopoietic stem and progenitor cells (HSPCs) are combined with a murine stromal cell line expressing a Notch ligand in a 3D cell aggregate. In ATOs, although only simple medium changes are required throughout the cultures, HSPCs differentiate into T cells with kinetics and phenotypes similar to those of endogenous thymopoiesis. This article describes protocols for the generation of ATOs from human and murine HSPCs. \u00a9 2022 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Expansion and preparation of MS5\u2010hDLL4 or MS5\u2010mDLL4 cells<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Isolation of human hematopoietic stem and progenitor cells (HSPCs; CD34+ cells)<\/jats:p><jats:p><jats:bold>Support Protocol 1<\/jats:bold>: Transduction of human HSPCs (CD34+ cells)<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Production of thymic progenitors and mature T cells from human HSPCs in artificial thymic organoids (ATOs)<\/jats:p><jats:p><jats:bold>Support Protocol 2<\/jats:bold>: Phenotype analysis of human ATO cells by flow cytometry<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: Isolation of murine HSPCs (Lin\u2010 Sca1+ cKit+; LSK) and hematopoietic stem cells (LSK CD150+ CD48\u2010)<\/jats:p><jats:p><jats:bold>Basic Protocol 5<\/jats:bold>: Production of thymic progenitors and mature T cells from murine HSPCs in ATOs<\/jats:p><jats:p><jats:bold>Support Protocol 3<\/jats:bold>: Phenotype analysis of murine ATO cells by flow cytometry<\/jats:p><jats:p><jats:bold>Alternate Protocol<\/jats:bold>: Generation of ATOs from single HSPCs<\/jats:p>","DOI":"10.1002\/cpz1.403","type":"journal-article","created":{"date-parts":[[2022,4,5]],"date-time":"2022-04-05T18:24:14Z","timestamp":1649183054000},"source":"Crossref","is-referenced-by-count":19,"title":["Generation of Artificial Thymic Organoids from Human and Murine Hematopoietic Stem and Progenitor Cells"],"prefix":"10.1002","volume":"2","author":[{"given":"Am\u00e9lie","family":"Montel\u2010Hagen","sequence":"first","affiliation":[{"name":"Department of Pathology and Laboratory Medicine, David Geffen School of Medicine University of California Los Angeles (UCLA)  Los Angeles 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