Series: | IN_VITRO DIFFERENTIATION SERIES |
Species: | Mouse (Mus musculus) |
Genomic View: | Zenbu |
Expression table: | FILE |
Link to TET: | TET |
Sample providers : | Hiroshi Kawamoto |
Germ layer: | mesoderm |
Primary cells or cell line: | primary cells |
Time span: | 6 days |
Number of time points: | 15 |
CollapseOverview
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T cells are produced in the thymus. The earliest T cell progenitors in the thymus are not fully committed to the T cell lineage but retain potentials to give rise to other lineage cells, myeloid cells, dendritic cells and natural killer cells. The T cell progenitors gradually lose their potential and commit to the T cell lineage through interacting with thymic epithelial cells. However, the exact mechanisms are still poorly understood. Especially, the transcriptional networks controlling the T cell fate determination remain elusive because of the lack of suitable experimental systems. Here, we have established a coculture system using EBF1KO hematopoietic progenitor cells (HPCs) with the TSt-4/Delta-like (DLL) 1 stromal cells that support the T cell differentiation[1,2]. By applying this time course samples to CAGE analysis, we examined the gene regulatory networks underlying the T cell lineage commitment from multipotent hematopoietic progenitors.
References: [1] Lin YC, Jhunjhunwala S, Benner C, Heinz S, Welinder E, Mansson R, Sigvardsson M, Hagman J, Espinoza CA, Dutkowski J, Ideker T, Glass CK, Murre C. A global network of transcription factors, involving E2A, EBF1 and Foxo1, that orchestrates B cell fate. Nat Immunol. 11(7): 635-643, 2010 [2] Ikawa T, Hirose S, Masuda K, Kakugawa K, Satoh R, Shibano-Satoh A, Kominami R,Katsura Y, and Kawamoto H. An essential developmental checkpoint for production of the T cell lineage. Science 329: 93-96, 2010
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ExpandSample description
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We used EBF1KO HPCs, which were isolated from day 15 fetal livers of EBF1KO mice[1]. EBF1KO HPCs were maintained on TSt-4 stromal cells in IMDM (SIGMA) supplemented with 10% FBS (Life Technologies), 2-ME (5 x 10-5 M; Nacalai tesque), streptomycin (100mg/ml), penicillin (100U/ml) (both from Life Technologies), SCF, IL-7 and Flt3-L (all from R&D). 1 x 106 EBF1KO cells were transferred to TSt-4/DLL1 cells to induce T lineage differentiation. T lineage commitment was verified by the surface expression of CD117 and CD25 at day 6 of the culture. After inducing T lineage differentiation, we harvested the samples at 0 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h of the culture. The 0 h time point serves as a control for the other time points (Figure 1).
Figure 1: Schematic experimental procedure for the time course analysis. EBF1KO cells were transferred and cultured on the TSt-4/DLL1 stromal cells to induce T cell differentiation. The cells were harvested at each time point indicated in the figure. Total RNA was purified using miRNeasy Micro Kit (Qiagen) and served for CAGE analysis.
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ExpandQuality control
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Figure 2: CAGE expression of marker genes in TPM.
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Profiled time course samples
Only samples that passed quality controls (Arner et al. 2015) are shown here. The entire set of samples are downloadable from FANTOM5 human / mouse samples