In Vitro Recapitulation of Murine Thymopoiesis from Single Hematopoietic Stem Cells

Montel‐Hagen, Amélie; Sun, Victoria; Casero, David; Tsai, Shau‐Wei; Zampieri, Alexandre; Jackson, Nicholas; Li, Suwen; Lopez, S. R.; Zhu, Yuhua; Chick, Brent; He, Chongbin; Barros, Stéphanie C. de; Seet, Christopher S.; Crooks, Gay M.

doi:10.1016/j.celrep.2020.108320

Cited by 25 publications

(42 citation statements)

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“…The generation of MS5-mDLL4 and MS5-hDLL4 cell lines in our lab has been previously described ( 27 , 29 ).…”

Section: Methodsmentioning

confidence: 99%

“…M-ATOs were generated as previously described ( 27 ). MS5-mDLL4 cells were harvested by trypsinization and resuspended in serum free M-ATO culture medium (“D/F12-B27”) composed of DMEM-F12 (Gibco, Cat# 11320033), 2% B27 supplement (ThermoFisher Scientific, Grand Island, NY, Cat# 17504-044), 30 µM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, St. Louis, MO, Cat# A8960-5G) reconstituted in 1X PBS, 1% penicillin/streptomycin (Gemini Bio-Products, West Sacramento, CA, Cat# 400-109), 1% Glutamax (ThermoFisher Scientific, Grand Island, NY, Cat# 35050-061), 5 ng/ml rmFLT3L (Peprotech, Rocky Hill, NJ, Cat# 250-31L), 5 ng/ml rmIL-7 (Peprotech, Cat# 217-17), 10 ng/ml rmSCF (Peprotech, Cat# 250-03) (of note SCF was added only for the first week of culture) and beta mercaptoethanol (bME) (0.05mM) (Sigma-Aldrich, Cat# M7522).…”

Section: Methodsmentioning

confidence: 99%

“…For all analyses DAPI + cells were gated out, and single cells were gated based on FSC-H vs. FSC-W and SSC-H vs. SSC-W. Gating strategies were performed as detailed in Supplementary Table 1 and presented in previous reports ( 27 , 28 ).…”

Section: Methodsmentioning

confidence: 99%

“…The thymopoiesis field has greatly benefited from in vitro models of T cell differentiation from HSPCs. One such model is the Artificial Thymic Organoid (ATO), a serum-free system that generates mature T cells from various HSPC sources, such as mouse bone marrow ( 27 ), human cord blood ( 28 ), and induced human pluripotent stem cells (iPSCs) ( 29 ). Considering the challenges of studying metabolic changes during in vivo human thymocyte development, in vitro models such as the ATO are potentially more tractable to perform metabolic measurements and perturbations.…”

Section: Introductionmentioning

confidence: 99%

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The Metabolic Landscape of Thymic T Cell Development In Vivo and In Vitro

et al. 2021

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Although metabolic pathways have been shown to control differentiation and activation in peripheral T cells, metabolic studies on thymic T cell development are still lacking, especially in human tissue. In this study, we use transcriptomics and extracellular flux analyses to investigate the metabolic profiles of primary thymic and in vitro-derived mouse and human thymocytes. Core metabolic pathways, specifically glycolysis and oxidative phosphorylation, undergo dramatic changes between the double-negative (DN), double-positive (DP), and mature single-positive (SP) stages in murine and human thymus. Remarkably, despite the absence of the complex multicellular thymic microenvironment, in vitro murine and human T cell development recapitulated the coordinated decrease in glycolytic and oxidative phosphorylation activity between the DN and DP stages seen in primary thymus. Moreover, by inducing in vitro T cell differentiation from Rag1-/- mouse bone marrow, we show that reduced metabolic activity at the DP stage is independent of TCR rearrangement. Thus, our findings suggest that highly conserved metabolic transitions are critical for thymic T cell development.

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“…The generation of MS5-mDLL4 and MS5-hDLL4 cell lines in our lab has been previously described ( 27 , 29 ).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

The Metabolic Landscape of Thymic T Cell Development In Vivo and In Vitro

et al. 2021

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“…M-ATO-mediated T cell development followed the kinetics and classical gene expression programs of in vivo T cell development and produced polyclonal mature CD4 + and CD8 + αβ T cells as well as T reg -like cells and γδ T cells. This system allowed for the efficient production of T-lineage progeny from single hematopoietic stem cells, thus allowing the assessment of developmental potential using single cells 29 .…”

Section: From Bedside To Benchmentioning

confidence: 99%

ThymUS in times of stress

Shissler

Bhandoola

2021

Nat Immunol

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The Global Thymus Network convened virtually for ThymUS 2020 on 3-4 November 2020. Participants shared recent advances in thymus biology and enjoyed a reprieve from 2020 stressors. Jonathan Sprent delivered the Charlie Surh Memorial Keynote at ThymUS 2020. Photographs of Jonathan Sprent and Charlie Surh individually (left and right, respectively) and together (center bottom). Charlie and Juan Carlos Zúñiga-Pflücker, who introduced the keynote, are pictured together at ICI/IUIS Melbourne 2016 (center top). Photographs were provided by Jonathan Sprent.

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Generation of Artificial Thymic Organoids from Human and Murine Hematopoietic Stem and Progenitor Cells

et al. 2022

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The generation of T cells is a complex, carefully orchestrated process that occurs in the thymus. The ability to mimic T cell differentiation in vitro has opened up avenues to better understand different stages of thymopoiesis but has also enabled the in vitro 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‐free 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. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expansion and preparation of MS5‐hDLL4 or MS5‐mDLL4 cells Basic Protocol 2: Isolation of human hematopoietic stem and progenitor cells (HSPCs; CD34+ cells) Support Protocol 1: Transduction of human HSPCs (CD34+ cells) Basic Protocol 3: Production of thymic progenitors and mature T cells from human HSPCs in artificial thymic organoids (ATOs) Support Protocol 2: Phenotype analysis of human ATO cells by flow cytometry Basic Protocol 4: Isolation of murine HSPCs (Lin‐ Sca1+ cKit+; LSK) and hematopoietic stem cells (LSK CD150+ CD48‐) Basic Protocol 5: Production of thymic progenitors and mature T cells from murine HSPCs in ATOs Support Protocol 3: Phenotype analysis of murine ATO cells by flow cytometry Alternate Protocol: Generation of ATOs from single HSPCs

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In Vitro Recapitulation of Murine Thymopoiesis from Single Hematopoietic Stem Cells

Cited by 25 publications

References 58 publications

The Metabolic Landscape of Thymic T Cell Development In Vivo and In Vitro

The Metabolic Landscape of Thymic T Cell Development In Vivo and In Vitro

ThymUS in times of stress

Generation of Artificial Thymic Organoids from Human and Murine Hematopoietic Stem and Progenitor Cells

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