{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T09:05:39Z","timestamp":1790759139827,"version":"4.1.0"},"reference-count":23,"publisher":"MDPI AG","issue":"19","license":[{"start":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T00:00:00Z","timestamp":1790467200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Key Research and Development Program of China grant","award":["2018YFA0108201"],"award-info":[{"award-number":["2018YFA0108201"]}]},{"name":"Research Starting Funding of South China University of Technology","award":["D6201880"],"award-info":[{"award-number":["D6201880"]}],"id":[{"id":"https:\/\/ror.org\/0530pts50","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Research Starting Funding of South China University of Technology","award":["K5180910"],"award-info":[{"award-number":["K5180910"]}],"id":[{"id":"https:\/\/ror.org\/0530pts50","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Research Starting Funding of South China University of Technology","award":["K5204120"],"award-info":[{"award-number":["K5204120"]}],"id":[{"id":"https:\/\/ror.org\/0530pts50","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Research Starting Funding of South China University of Technology","award":["D6212440"],"award-info":[{"award-number":["D6212440"]}],"id":[{"id":"https:\/\/ror.org\/0530pts50","id-type":"ROR","asserted-by":"crossref"}]},{"award":["D6201880"],"award-info":[{"award-number":["D6201880"]}],"id":[{"id":"https:\/\/ror.org\/0530pts50","id-type":"ROR","asserted-by":"publisher"}]},{"award":["K5180910"],"award-info":[{"award-number":["K5180910"]}],"id":[{"id":"https:\/\/ror.org\/0530pts50","id-type":"ROR","asserted-by":"publisher"}]},{"award":["K5204120"],"award-info":[{"award-number":["K5204120"]}],"id":[{"id":"https:\/\/ror.org\/0530pts50","id-type":"ROR","asserted-by":"publisher"}]},{"award":["D6212440"],"award-info":[{"award-number":["D6212440"]}],"id":[{"id":"https:\/\/ror.org\/0530pts50","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Research Starting Funding of the Second Affiliated Hospital of South China University of Technology","award":["KY09060026"],"award-info":[{"award-number":["KY09060026"]}]},{"name":"Research Starting Funding of the Second Affiliated Hospital of South China University of Technology","award":["KY09090002"],"award-info":[{"award-number":["KY09090002"]}]},{"name":"Research Agreement between South China University of Technology and Guangzhou First People\u2019s Hospital","award":["D9194290"],"award-info":[{"award-number":["D9194290"]}],"id":[{"id":"https:\/\/ror.org\/0530pts50","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Thymic epithelial cells (TECs) provide essential signals for T-cell development, yet the mechanisms governing human TEC differentiation and the acquisition of T-cell-supporting function remain incompletely understood. Using a three-stage system that directs human embryonic stem cells (hESCs) through definitive endoderm and third pharyngeal pouch endoderm (3PPE) toward a thymic epithelial-like lineage, we investigated the role of HOXA3 during thymic epithelial-like cell (TEC-like cell) differentiation. HOXA3 knockdown during 3PPE-to-TEC-like cell transition impaired thymic epithelial differentiation, as indicated by reduced K8, AIRE, and CD205 expression and decreased FOXN1 protein. HOXA3-knockdown TEC-like cells also showed a markedly reduced capacity to support the generation of CD45+CD3+ cells from cord blood CD34+ progenitors. Bulk RNA sequencing identified 1040 upregulated and 774 downregulated genes, revealing broad remodeling of cell-cycle, motility, chemotaxis, angiogenesis, and tissue-remodeling programs. CXCL12 was among the downregulated genes, and its reduction was confirmed at both mRNA and protein levels. Motif analysis identified a putative HOXA3-binding site, and conventional ChIP-PCR showed enrichment of the predicted CXCL12 regulatory region in HOXA3 immunoprecipitates. Recombinant CXCL12 significantly increased CD45+CD3+ cell generation in HOXA3-knockdown co-cultures. Together, these data support an association between HOXA3 knockdown, reduced acquisition of a thymic epithelial-like phenotype, and diminished early T-lineage support, with CXCL12 partially accounting for the functional phenotype. This study provides a tractable hESC-based model for studying human thymic epithelial development and TEC-mediated early T-lineage differentiation.<\/jats:p>","DOI":"10.3390\/cells15191764","type":"journal-article","created":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T08:18:46Z","timestamp":1790583526000},"page":"1764","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["HOXA3 Knockdown Impairs hESC-Derived Thymic Epithelial-like Cell Differentiation and CXCL12-Associated Early T-Lineage Support"],"prefix":"10.3390","volume":"15","author":[{"given":"Huanhuan","family":"Shan","sequence":"first","affiliation":[{"name":"Laboratory of Stem Cells and Translational Medicine, Center for Medical Research on Innovation and Translation, Institute of Clinical Medicine, The Second Affiliated Hospital, School of Medicine, South China University of Technology, Guangzhou 510006, China"},{"name":"Laboratory of Stem Cells and Translational Medicine, Institute for Life Science, School of Medicine, South China University of Technology, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Yingjie","family":"Fu","sequence":"additional","affiliation":[{"name":"Laboratory of Stem Cells and Translational Medicine, Center for Medical Research on Innovation and Translation, Institute of Clinical Medicine, The Second Affiliated Hospital, School of Medicine, South China University of Technology, Guangzhou 510006, China"},{"name":"Laboratory of Stem Cells and Translational Medicine, Institute for Life Science, School of Medicine, South China University of Technology, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Xinyi","family":"Shi","sequence":"additional","affiliation":[{"name":"Laboratory of Stem Cells and Translational Medicine, Center for Medical Research on Innovation and Translation, Institute of Clinical Medicine, The Second Affiliated Hospital, School of Medicine, South China University of Technology, Guangzhou 510006, China"},{"name":"Laboratory of Stem Cells and Translational Medicine, Institute for Life Science, School of Medicine, South China University of Technology, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"data-curation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"}]},{"given":"Sen","family":"Chen","sequence":"additional","affiliation":[{"name":"Laboratory of Stem Cells and Translational Medicine, Center for Medical Research on Innovation and Translation, Institute of Clinical Medicine, The Second Affiliated Hospital, School of Medicine, South China University of Technology, Guangzhou 510006, China"},{"name":"Laboratory of Stem Cells and Translational Medicine, Institute for Life Science, School of Medicine, South China University of Technology, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1402-7402","authenticated-orcid":false,"given":"Yuyou","family":"Duan","sequence":"additional","affiliation":[{"name":"Laboratory of Stem Cells and Translational Medicine, Center for Medical Research on Innovation and Translation, Institute of Clinical Medicine, The Second Affiliated Hospital, School of Medicine, South China University of Technology, Guangzhou 510006, China"},{"name":"Laboratory of Stem Cells and Translational Medicine, Institute for Life Science, School of Medicine, South China University of Technology, Guangzhou 510006, China"},{"name":"National Engineering Research Center for Tissue Restoration and Reconstruction, South China University of Technology, Guangzhou 510006, China"},{"name":"The Innovation Centre of Ministry of Education for Development and Diseases, The Second Affiliated Hospital, School of Medicine, South China University of Technology, Guangzhou 510006, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Lopes, N., Serg\u00e9, A., Ferrier, P., and Irla, M. (2015). Thymic Crosstalk Coordinates Medulla Organization and T-Cell Tolerance Induction. Front. Immunol., 6.","DOI":"10.3389\/fimmu.2015.00365"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1755","DOI":"10.1016\/j.jaci.2021.09.038","article-title":"Human stem cell-derived thymic epithelial cells enhance human T-cell development in a xenogeneic thymus","volume":"149","author":"Danzl","year":"2022","journal-title":"J. Allergy Clin. Immunol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"210","DOI":"10.1111\/imm.70160","article-title":"Thymic APC Networks Orchestrate T-Cell Selection: Mechanisms and Therapeutic Opportunities in Immune Disorders","volume":"179","author":"Luo","year":"2026","journal-title":"Immunology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1038\/nri3667","article-title":"Positive and negative selection of the T cell repertoire: What thymocytes see (and don\u2019t see)","volume":"14","author":"Klein","year":"2014","journal-title":"Nat. Rev. Immunol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"eaeb2970","DOI":"10.1126\/sciadv.aeb2970","article-title":"Age-related thymic involution: Mechanistic insights and rejuvenating approaches to restore immune function","volume":"12","author":"Santamaria","year":"2026","journal-title":"Sci. Adv."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Palmer, D.B. (2013). The Effect of Age on Thymic Function. Front. Immunol., 4.","DOI":"10.3389\/fimmu.2013.00316"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"101732","DOI":"10.1016\/j.smim.2023.101732","article-title":"Human thymus in health and disease: Recent advances in diagnosis and biology","volume":"66","author":"Bosticardo","year":"2023","journal-title":"Semin. Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/j.stem.2013.04.004","article-title":"Generation of Functional Thymic Epithelium from Human Embryonic Stem Cells that Supports Host T Cell Development","volume":"13","author":"Parent","year":"2013","journal-title":"Cell Stem Cell"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1016\/j.stem.2013.06.014","article-title":"Directed Differentiation of Human Embryonic Stem Cells into Thymic Epithelial Progenitor-like Cells Reconstitutes the Thymic Microenvironment In Vivo","volume":"13","author":"Sun","year":"2013","journal-title":"Cell Stem Cell"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"925","DOI":"10.1016\/j.stemcr.2014.04.009","article-title":"FOXN1GFP\/w Reporter hESCs Enable Identification of Integrin-\u03b24, HLA-DR, and EpCAM as Markers of Human PSC-Derived FOXN1+ Thymic Epithelial Progenitors","volume":"2","author":"Soh","year":"2014","journal-title":"Stem Cell Rep."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3865","DOI":"10.1242\/dev.059998","article-title":"Mechanisms of Thymus Organogenesis and Morphogenesis","volume":"138","author":"Gordon","year":"2011","journal-title":"Development"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Provin, N., and Giraud, M. (2022). Differentiation of Pluripotent Stem Cells into Thymic Epithelial Cells and Generation of Thymic Organoids: Applications for Therapeutic Strategies Against APECED. Front. Immunol., 13.","DOI":"10.3389\/fimmu.2022.930963"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1989","DOI":"10.1242\/dev.121.7.1989","article-title":"The Role of Hoxa-3 in Mouse Thymus and Thyroid Development","volume":"121","author":"Manley","year":"1995","journal-title":"Development"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3697","DOI":"10.1242\/dev.110833","article-title":"Multiple Roles for HOXA3 in Regulating Thymus and Parathyroid Differentiation and Morphogenesis in Mouse","volume":"141","author":"Chojnowski","year":"2014","journal-title":"Development"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"316","DOI":"10.1006\/dbio.2001.0342","article-title":"Hoxa3 and Pax1 Regulate Epithelial Cell Death and Proliferation during Thymus and Parathyroid Organogenesis","volume":"236","author":"Su","year":"2001","journal-title":"Dev. Biol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Fu, Y., Zhang, X., Wu, H., Zhang, P., Liu, S., Guo, T., Shan, H., Liang, Y., Chen, H., and Xie, J. (2024). HOXA3 Functions as the On-off Switch to Regulate the Development of hESC-Derived Third Pharyngeal Pouch Endoderm Through EPHB2-Mediated Wnt Pathway. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1258074"},{"key":"ref_17","unstructured":"Liston, A. (2010). Transcriptional Regulation of Thymus Organogenesis and Thymic Epithelial Cell Differentiation. Progress in Molecular Biology and Translational Science, Academic Press."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1038\/35095500","article-title":"Lymphostromal Interactions in Thymic Development and Function","volume":"1","author":"Anderson","year":"2001","journal-title":"Nat. Rev. Immunol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"4521","DOI":"10.4049\/jimmunol.171.9.4521","article-title":"Critical Role for CXCR4 Signaling in Progenitor Localization and T Cell Differentiation in the Postnatal Thymus","volume":"171","author":"Plotkin","year":"2003","journal-title":"J. Immunol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"4649","DOI":"10.4049\/jimmunol.170.9.4649","article-title":"A Role of CXC Chemokine Ligand 12\/Stromal Cell-Derived Factor-1\/Pre-B Cell Growth Stimulating Factor and Its Receptor CXCR4 in Fetal and Adult T Cell Development in Vivo","volume":"170","author":"Ara","year":"2003","journal-title":"J. Immunol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Romano, R., Palamaro, L., Fusco, A., Giardino, G., Gallo, V., Del Vecchio, L., and Pignata, C. (2013). FOXN1: A Master Regulator Gene of Thymic Epithelial Development Program. Front. Immunol., 4.","DOI":"10.3389\/fimmu.2013.00187"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"571","DOI":"10.1146\/annurev.immunol.23.021704.115601","article-title":"A Central Role for Central Tolerance","volume":"24","author":"Kyewski","year":"2006","journal-title":"Annu. Rev. Immunol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"5742","DOI":"10.1073\/pnas.93.12.5742","article-title":"The nu Gene Acts Cell-Autonomously and is Required for Differentiation of Thymic Epithelial Progenitors","volume":"93","author":"Blackburn","year":"1996","journal-title":"Proc. Natl. Acad. Sci. USA"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/19\/1764\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T08:34:03Z","timestamp":1790757243000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/19\/1764"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,27]]},"references-count":23,"journal-issue":{"issue":"19","published-online":{"date-parts":[[2026,10]]}},"alternative-id":["cells15191764"],"URL":"https:\/\/doi.org\/10.3390\/cells15191764","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,27]]}}}