T-cell differentiation

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

Overview

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

Sample description

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.

Quality control
Figure 2: CAGE expression of marker genes in TPM.

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

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12971-138H1	TSt-4/DLL1 feeder cells, biol_rep1	{{{time}}}	{{{donor}}}
12972-138H2	EBF KO HPCs induced to T cell	00hr00min	biol_rep1
12973-138H3	EBF KO HPCs induced to T cell	00hr30min	biol_rep1
12974-138H4	EBF KO HPCs induced to T cell	01hr	biol_rep1
12975-138H5	EBF KO HPCs induced to T cell	02hr	biol_rep1
12976-138H6	EBF KO HPCs induced to T cell	04hr	biol_rep1
12977-138H7	EBF KO HPCs induced to T cell	06hr	biol_rep1
12978-138H8	EBF KO HPCs induced to T cell	08hr	biol_rep1
12979-138H9	EBF KO HPCs induced to T cell	10hr	biol_rep1
12980-138I1	EBF KO HPCs induced to T cell	12hr	biol_rep1
12981-138I2	EBF KO HPCs induced to T cell	24hr	biol_rep1
12982-138I3	EBF KO HPCs induced to T cell	day02	biol_rep1
12983-138I4	EBF KO HPCs induced to T cell	day03	biol_rep1
12984-138I5	EBF KO HPCs induced to T cell	day04	biol_rep1
12985-138I6	EBF KO HPCs induced to T cell	day05	biol_rep1
12986-138I7	EBF KO HPCs induced to T cell	day06	biol_rep1
12987-138I8	TSt-4/DLL1 feeder cells, biol_rep2	{{{time}}}	{{{donor}}}
12988-138I9	EBF KO HPCs induced to T cell	00hr00min	biol_rep2
12989-139A1	EBF KO HPCs induced to T cell	00hr30min	biol_rep2
12990-139A2	EBF KO HPCs induced to T cell	01hr	biol_rep2
12991-139A3	EBF KO HPCs induced to T cell, 02hr, biol_rep2	{{{time}}}	{{{donor}}}
12992-139A4	EBF KO HPCs induced to T cell	04hr	biol_rep2
12993-139A5	EBF KO HPCs induced to T cell	06hr	biol_rep2
12994-139A6	EBF KO HPCs induced to T cell	08hr	biol_rep2
12995-139A7	EBF KO HPCs induced to T cell	10hr	biol_rep2
12996-139A8	EBF KO HPCs induced to T cell	12hr	biol_rep2
12997-139A9	EBF KO HPCs induced to T cell	24hr	biol_rep2
12998-139B1	EBF KO HPCs induced to T cell	day02	biol_rep2
12999-139B2	EBF KO HPCs induced to T cell	day03	biol_rep2
13000-139B3	EBF KO HPCs induced to T cell	day04	biol_rep2
13001-139B4	EBF KO HPCs induced to T cell	day05	biol_rep2
13002-139B5	EBF KO HPCs induced to T cell	day06	biol_rep2
13003-139B6	TSt-4/DLL1 feeder cells, biol_rep3	{{{time}}}	{{{donor}}}
13004-139B7	EBF KO HPCs induced to T cell	00hr00min	biol_rep3
13005-139B8	EBF KO HPCs induced to T cell	00hr30min	biol_rep3
13006-139B9	EBF KO HPCs induced to T cell	01hr	biol_rep3
13007-139C1	EBF KO HPCs induced to T cell	02hr	biol_rep3
13008-139C2	EBF KO HPCs induced to T cell	04hr	biol_rep3
13009-139C3	EBF KO HPCs induced to T cell	06hr	biol_rep3
13010-139C4	EBF KO HPCs induced to T cell	08hr	biol_rep3
13011-139C5	EBF KO HPCs induced to T cell	10hr	biol_rep3
13012-139C6	EBF KO HPCs induced to T cell	12hr	biol_rep3
13013-139C7	EBF KO HPCs induced to T cell, 24hr, biol_rep3	{{{time}}}	{{{donor}}}
13014-139C8	EBF KO HPCs induced to T cell	day02	biol_rep3
13015-139C9	EBF KO HPCs induced to T cell	day03	biol_rep3
13016-139D1	EBF KO HPCs induced to T cell	day04	biol_rep3
13017-139D2	EBF KO HPCs induced to T cell	day05	biol_rep3
13018-139D3	EBF KO HPCs induced to T cell, day06, biol_rep3	{{{time}}}	{{{donor}}}