Abstract
The thymus educates thymocytes through a selection process mediated by thymic epithelial cells (TECs). Recent advances have made the generation of T lymphocytes from induced pluripotent stem cells (iPSc) a promising therapeutic strategy. However, current approaches often fail to replicate the thymic niche, leading to impaired T cell generation. Here we address the production of functional mature iPSc-derived TECs supporting in vitro T cell generation. We optimize thymic lineage differentiation through an unbiased multifactorial experimental design. By modulating specific signaling pathways, we generate progenitors that mature into medullary and cortical TECs. Co-culture with primary hematopoietic progenitors in a 3D thymic organoid setup induces their differentiation into CD4+ and CD8+ T cells. Importantly, thymic organoids support multilineage differentiation, with dendritic cell populations also emerging. Thus, the presented thymic organoid model provides a practical platform for studying thymic cellular interactions and thymopoiesis in vitro, and opens further research perspectives towards cell-based therapies.
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Data availability
The DGE-seq RNA sequencing data related to the DOE-based factor screening and generated in this study have been deposited in the GEO database under accession code GSE302942. The DGE-seq data related to the iPSc to TEP differentiation and generated in this study have been deposited in the GEO database under the accession code GSE304598. The CD205 TEP RNA-seq data generated in this study have been deposited in the GEO database under accession code GSE294118. The D7 hTO TEC scRNA-seq data generated in this study have been deposited in the GEO database under accession code GSE293928. The RNA-seq data of HLA-DRhi, HLA-DRlo TECs and thymic DCs in D28 hTOs generated in this study have been deposited in the GEO database under accession code GSE294061. The scRNA-seq data of D28 hTO detaching cells generated in this study have been deposited in the GEO database under accession code GSE293930. The scRNA-seq data of early pharyngeal development (Han) reused in this study are available in the GEO database under accession code GSE136689. The scRNA-seq data of late pharyngeal development (Magaletta) reused in this study are available in the GEO database under accession code GSE182135. Park’s scRNA-seq data of human thymic cells reused in this study are available at [https://cellatlas.io/studies/thymus-development#resources]. Bautista’s scRNA-seq data of human TECs reused in this study are available in the GEO database under accession code GSE147520. RNA-seq data from human mTEChi, mTEClo, and cTEC populations, used as controls in this study, are available in the GEO database under accession code GSE176445. All other data are available in the article and its Supplementary files or from the corresponding author upon request. Source data are provided with this paper.
Code availability
We have provided the documented R code that we developed for the calculation of gene tissue specificity scores93 as a Git repository available from https://github.com/TeamGiraud/GeneTissueSpe and as a Shiny application at https://nathanprovin.shinyapps.io/GeneTissueSpe.
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Acknowledgements
This work was supported by the EJP-Rare Disease JTC2019 program TARID project (ANR-19-RAR4-0011-05), “la Région Pays de la Loire” through the RFI Bioregate grant (ThymIPS), the NORD (The APS Type 1 Foundation grant) ID: 22001 and the “SATT Ouest Valorisation” project OrgaTreg to M.G., as well as “la Fondation pour la Recherche Médicale” (FRM) under grant Equipe FRM EQU202303016266 to C.G. and M.G. This work was also partially supported by the ANR SelfExpress (ANR-22-CE15-0045-01) and by "la Région Pays de la Loire" through the Trajectoire Nationale program (2023_01170) to M.G. N.P. was supported by “la Fondation d’entreprise ProGreffe”. M.d.A. received PhD fellowship support from the FRM (grant no. 17366). This work was partially funded by the Labex IGOprogram supported by the National Research Agency via the investment program ANR-11-LABX-0016-01. We thank the members of the “Genomic’IC” core facility headed by Franck Letourneur at Cochin Institute, Paris, France, for bulk RNA-seq data production, as well as, Drs Pärt Peterson, Julia Maslovskaja and Kai Kisand from Tartu University, Estonia, for human TEC RNA-seq datasets. We thank the iPSC and the MicroPICell core facilities of Nantes supported by IBiSA and Biogenouest, for the use of their resources and technical support. We specifically thank Caroline Chariau and Isabelle Leray (iPSC facility), as well as Anne Gaignerie (MicroPICell) for her advice on iPSc-to-HSPC differentiation, and Laurence Delbos for her advice on flow cytometry. We also thank Ella Beaumann for bioinformatics assistance and Léana Lecompte and Teresa Rodriguez-Trenchs for their help in iPSc culture and differentiation.
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N.P. and M.G. designed the study and wrote the manuscript; N.P., M.d.A. performed most of the experimental work; A.L.B. performed key HSPC experiments; A.B., L.B., C.F., J.P., and P.H. performed experiments; N.P., E.K., and M.G. performed bioinformatics analyses; P.M., O.B., and X.S. provided key material; C.G. and L.D. contributed to the study design and edited the manuscript.
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Provin, N., d’Arco, M., Le Bozec, A. et al. Combinatory differentiation of human induced pluripotent stem cells generates functional thymic epithelium driving dendritic cell and CD4/CD8 T cell development. Nat Commun (2026). https://doi.org/10.1038/s41467-026-68675-y
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DOI: https://doi.org/10.1038/s41467-026-68675-y
