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

Seet, Christopher S. and He, Chongbin and Bethune, Michael T. and Li, Suwen and Chick, Brent and Gschweng, Eric H. and Zhu, Yuhua and Kim, Kenneth and Kohn, Donald B. and Baltimore, David and Crooks, Gay M. and Montel-Hagen, Amélie (2017) Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids. Nature Methods, 14 (5). pp. 521-530. ISSN 1548-7091. PMCID PMC5426913.

[img] PDF - Accepted Version
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[img] Image (JPEG) (Supplementary Figure 1: ATOs form solid tissue-like structures) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 2: The starting number of HSPCs per ATO affects cell yield per HSPC but not the total cell output or T cell differentiation) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 3: T cell differentiation in ATOs is highly reproducible and not affected by B27 lot variation, xeno-free B27 or stromal irradiation) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 4: Enhanced T cell positive selection and maturation in ATOs compared with OP9-DL1 monolayer co-cultures) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 5: Enhanced positive selection in ATOs requires 3D structure and optimal cell line and culture medium) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 6: Recapitulation of thymopoiesis and naive T cell phenotype in ATOs) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 7: Generation of T cells from multiple HSPC sources and subsets) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 8: TCR diversity and functional validation of ATO-derived CD4^+ T cells) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 9: Differentiation and allelic exclusion of TCR-engineered T cells in ATOs) - Supplemental Material
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[img] PDF (Supplementary Figures 1–9) - Supplemental Material
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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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Baltimore, David0000-0001-8723-8190
Alternate Title:Highly efficient generation of conventional and engineered mature T cells from human hematopoietic stem and progenitor cells using artificial thymic organoids
Additional Information:© 2017 Macmillan Publishers Limited. Received 02 December 2016. Accepted 03 March 2017. Published online 03 April 2017. 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. These authors contributed equally to this work. Gay M Crooks & Amélie Montel-Hagen. Author Contributions: 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 financial 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. Data availability. The data that support the findings of this study are available from the corresponding author upon request. Sequences for the TCR-encoding genes from this study are available in the NCBI Gene Expression Omnibus repository under accession number GSE94968.
Group:Millard and Muriel Jacobs Genetics and Genomics Laboratory
Funding AgencyGrant Number
NIHR01 AG049753
NIHP01 HL073104
NIH Predoctoral FellowshipT32HL066992
Tower Cancer Research FoundationUNSPECIFIED
Broad Stem Cell Research CenterUNSPECIFIED
Prostate Cancer Foundation15CHAL02
Jane Coffin Childs Memorial Fund for Medical ResearchUNSPECIFIED
NIH5P30 AI028697
Millard and Muriel Jacobs Genetics and Genomics LaboratoryUNSPECIFIED
Issue or Number:5
PubMed Central ID:PMC5426913
Record Number:CaltechAUTHORS:20170124-111140333
Persistent URL:
Official Citation:Generation of mature T cells from human hematopoietic stem and progenitor cells in artificial thymic organoids Christopher S Seet, Chongbin He, Michael T Bethune, Suwen Li, Brent Chick, Eric H Gschweng, Yuhua Zhu, Kenneth Kim, Donald B Kohn, David Baltimore, Gay M Crooks & Amélie Montel-Hagen Nature Methods (2017) doi:10.1038/nmeth.4237
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:73667
Deposited By: George Porter
Deposited On:03 Apr 2017 21:44
Last Modified:21 Apr 2020 17:42

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