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Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids

Nature Methods volume 14, pages 521530 (2017) | Download Citation


Studies of human T cell development require robust model systems that recapitulate the full span of thymopoiesis, from hematopoietic stem and progenitor cells (HSPCs) through to mature T cells. Existing in vitro models induce T cell commitment from human HSPCs; however, differentiation into mature CD3+TCR-αβ+ single-positive CD8+ or CD4+ cells is limited. We describe here a serum-free, artificial thymic organoid (ATO) system that supports efficient and reproducible in vitro differentiation and positive selection of conventional human T cells from all sources of HSPCs. ATO-derived T cells exhibited mature naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function. ATOs initiated with TCR-engineered HSPCs produced T cells with antigen-specific cytotoxicity and near-complete lack of endogenous TCR Vβ expression, consistent with allelic exclusion of Vβ-encoding loci. ATOs provide a robust tool for studying human T cell differentiation and for the future development of stem-cell-based engineered T cell therapies.

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We thank J. Scholes and F. Codrea at the UCLA Broad Stem Cell Research Center (BSCRC) Flow Cytometry Core for assistance with FACS sorting, R. Chan for assistance with specimen processing, C. Parekh (Children's Hospital Los Angeles) for generous assistance with thymus samples, M. Sehl (UCLA) for assistance with MPB collection, and A. Cooper (UCLA) for helpful advice and discussion. We thank I. Antoshechkin (Millard and Muriel Jacobs Genetics and Genomics Laboratory, Caltech), who developed the method for, and who assisted with, TCR sequencing analysis, A. Ribas (UCLA) for the NY-ESO-1 and MART-1 TCR constructs, J. Zuniger-Pflucker (University of Toronto) for OP9-DL1 cells, L. Coulombel for MS-5 cells and J. Chute (UCLA) for U266 cells. This work was supported by the NIH (grants R01 AG049753 (G.M.C.), 1R21AI119927 (G.M.C. and A.M.-H.), P01 HL073104 (G.M.C. and D.B.K.) and T32HL066992 (C.S.S.)), the Tower Cancer Research Foundation (C.S.S.), a UCLA BSCRC Innovation award (G.M.C. and D.B.K.) and a BSCRC Clinical Fellowship (C.S.S.). M.T.B. and D.B. are supported by Prostate Cancer Foundation Challenge Award 15CHAL02, and M.T.B. is the recipient of a Jane Coffin Childs Postdoctoral Fellowship. Core services were supported by the UCLA Jonsson Comprehensive Cancer Center shared facility (TPCL, grant 5P30CA016042), the UCLA Immunogenetics Center, the UCLA Center for AIDS Research Virology Core Lab and the UCLA AIDS Institute (grant 5P30 AI028697), and the Millard and Muriel Jacobs Genetics and Genomics Laboratory at Caltech.

Author information

Author notes

    • Gay M Crooks
    •  & Amélie Montel-Hagen

    These authors contributed equally to this work.


  1. Division of Hematology–Oncology, Department of Medicine, David Geffen School of Medicine (DGSOM), University of California Los Angeles (UCLA), Los Angeles, California, USA.

    • Christopher S Seet
  2. Department of Pathology and Laboratory Medicine, DGSOM, UCLA, Los Angeles, California, USA.

    • Chongbin He
    • , Suwen Li
    • , Brent Chick
    • , Yuhua Zhu
    • , Kenneth Kim
    • , Gay M Crooks
    •  & Amélie Montel-Hagen
  3. Division of Biology and Biological Engineering, California Institute of Technology (Caltech), Pasadena, California, USA.

    • Michael T Bethune
    •  & David Baltimore
  4. Department of Microbiology, Immunology and Molecular Genetics, DGSOM, UCLA, Los Angeles, California, USA.

    • Eric H Gschweng
    •  & Donald B Kohn
  5. Division of Pediatric Hematology–Oncology, Department of Pediatrics, DGSOM, UCLA, Los Angeles, California, USA.

    • Donald B Kohn
    •  & Gay M Crooks
  6. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, California, USA.

    • Donald B Kohn
    •  & Gay M Crooks
  7. Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, California, USA.

    • Donald B Kohn
    •  & Gay M Crooks


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C.S.S. and A.M.-H. designed and performed experiments, analyzed data, prepared figures and co-wrote the manuscript; C.H. performed histological experiments and, with B.C., assisted with in vivo experiments; S.L. assisted with ATO analysis and T cell functional assays; K.K. assisted with ATO cultures; Y.Z. performed human specimen processing and cultured the cell lines; E.H.G. and D.B.K. provided critical reagents and conceptual advice and edited the manuscript; M.T.B. and D.B. devised the approach for, and performed, TCR repertoire sequencing analysis and provided critical reagents; and G.M.C. and A.M.-H. co-directed the project and co-wrote the manuscript.

Competing interests

Kite Pharma, Inc. is supporting the preclinical research of the ATO system at UCLA with G.M.C. as principal investigator. Kite Pharma, Inc. also holds an exclusive license to certain intellectual property that relates to the ATO system.

Corresponding author

Correspondence to Gay M Crooks.

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