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Enumeration of benzidine positive cells, as an indication of hemoglobin synthesis, was carried out to monitor differentiation. The time course of Epo-induced differentiation of J2E cells shows that hemoglobin production increases markedly 24-48h after stimulation (Figure 1).<br>
Enumeration of benzidine positive cells, as an indication of hemoglobin synthesis, was carried out to monitor differentiation. The time course of Epo-induced differentiation of J2E cells shows that hemoglobin production increases markedly 24-48h after stimulation (Figure 1).<br>
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<br>
<html><img src='https://fantom5-collaboration.gsc.riken.jp/resource_browser/images/TC_qc/J2E_Fig1.png'></html>
<html><img src='https://fantom5-collaboration.gsc.riken.jp/resource_browser/images/TC_qc/J2E_Fig1.png'></html><br>
Figure 1. Benzidine positive cells were enumerated at each time point. Three biological replicates were analysed.<br>
Figure 1. Benzidine positive cells were enumerated at each time point. Three biological replicates were analysed.<br>
|TCQuality_control=Expression of the following genes was assessed to determine the validity of this cell line as a model of Epo-induced erythroid differentiation (Figure 2). All these genes are required for normal erythroid differentiation.<br>
|TCQuality_control=Expression of the following genes was assessed to determine the validity of this cell line as a model of Epo-induced erythroid differentiation (Figure 2). All these genes are required for normal erythroid differentiation.<br>
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<html><img src='https://fantom5-collaboration.gsc.riken.jp/resource_browser/images/TC_qc/J2E_Fig2.png' onclick='javascript:window.open("https://fantom5-collaboration.gsc.riken.jp/resource_browser/images/TC_qc/J2E_Fig2.png", "imgwindow", "width=768,height=785");' style='width:700px;cursor:pointer'/></html>
<html><img src='https://fantom5-collaboration.gsc.riken.jp/resource_browser/images/TC_qc/J2E_Fig2.png' onclick='javascript:window.open("https://fantom5-collaboration.gsc.riken.jp/resource_browser/images/TC_qc/J2E_Fig2.png", "imgwindow", "width=768,height=785");' style='width:700px;cursor:pointer'/></html>
Figure 2. Expression of key genes associated with erythroid differentiation. TPM: Tags per million.<br>
Figure 2. Expression of key genes associated with erythroid differentiation. TPM: Tags per million.<br>
<br>
Refereneces:<br>
[6] Klinken SP, Nicola NA, Johnson GR. In vitro-derived leukemic erythroid cell lines induced by a raf- and myc-containing retrovirus differentiate in response to erythropoietin. Proc Natl Acad Sci U S A 1988; 85: 8506-8510.<br>
[7] Tilbrook PA, Bittorf T, Callus BA, Busfield SJ, Ingley E, Klinken SP. Regulation of the erythropoietin receptor and involvement of JAK2 in differentiation of J2E erythroid cells. Cell Growth Differ 1996; 7: 511-520.<br>
[8] Tilbrook PA, Ingley E, Williams JH, Hibbs ML, Klinken SP. Lyn tyrosine kinase is essential for erythropoietin-induced differentiation of J2E erythroid cells. EMBO J 1997; 16: 1610-1619.<br>
[9] Busfield SJ, Klinken SP. Erythropoietin-induced stimulation of differentiation and proliferation in J2E cells is not mimicked by chemical induction. Blood 1992; 80: 412-419.<br>
[10] Lodish HF, Hilton DJ, Klingmuller U, Watowich SS, Wu H. The erythropoietin receptor: biogenesis, dimerization, and intracellular signal transduction. Cold Spring Harb Symp Quant Biol 1995; 60: 93-104.<br>
[11] Meguro K, Igarashi K, Yamamoto M, Fujita H, Sassa S. The role of the erythroid-specific delta-aminolevulinate synthase gene expression in erythroid heme synthesis. Blood 1995; 86: 940-948.<br>
[12] Stamatoyannopoulos G. Control of globin gene expression during development and erythroid differentiation. Exp Hematol 2005; 33: 259-271.<br>
[13] Tsai SF, Martin DI, Zon LI, D'Andrea AD, Wong GG, Orkin SH. Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells. Nature 1989; 339: 446-451.<br>
[14] Whitelaw E, Tsai SF, Hogben P, Orkin SH. Regulated expression of globin chains and the erythroid transcription factor GATA-1 during erythropoiesis in the developing mouse. Mol Cell Biol 1990; 10: 6596-6606.<br>
[15] Miller IJ, Bieker JJ. A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Kruppel family of nuclear proteins. Mol Cell Biol 1993; 13: 2776-2786.<br>
[16] Andrews NC, Erdjument-Bromage H, Davidson MB, Tempst P, Orkin SH. Erythroid transcription factor NF-E2 is a haematopoietic-specific basic- leucine zipper protein. Nature 1993; 362: 722-728.<br>
}}
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Revision as of 14:34, 11 December 2014

Series:IN_VITRO DIFFERENTIATION SERIES
Species:Mouse (Mus musculus)
Genomic View:Zenbu
Expression table:[{{{tet_config}}} FILE]
Link to TET:[{{{tet_file}}} TET]
Sample providers :Peter Klinken
Germ layer:{{{germ_layer}}}
Primary cells or cell line:{{{primary_cells}}}
Time span:{{{time_span}}}
Number of time points:{{{number_time_points}}}


Overview

Erythropoietin (Epo) is the hormone, which regulates red blood cell production1. It is produced primarily in the kidney, and binds to Epo receptors (Epor) on the surface of immature erythroid cells in the bone marrow, thereby initiating the final stages of red cell maturation[1,2]. Following binding of Epo to its cognate receptor, a series of intracellular signaling cascades are activated, including stimulation of the JAK/STAT and ras/MAP kinase pathways[3,4]. This leads to enhanced cell division, followed by terminal differentiation which is characterized by the production of hemoglobin. In addition, morphological changes occur involving a reduction in cell size, nuclear condensation, and eventually extrusion of the nucleus to produce reticulocytes. Mature red blood cells (erythrocytes) containing large amounts of hemoglobin then circulate around the body transporting oxygen and carbon dioxide [5].

References:
[1] Bunn HF. Erythropoietin. Cold Spring Harbor perspectives in medicine 2013; 3: a011619.
[2] Koury MJ, Koury ST, Bondurant MC, Graber SE. Correlation of the molecular and anatomical aspects of renal erythropoietin production. Contributions to nephrology 1989; 76: 24-29; discussion 30-22.
[3] Richmond TD, Chohan M, Barber DL. Turning cells red: signal transduction mediated by erythropoietin. Trends Cell Biol 2005; 15: 146-155.
[4] Ingley E. Integrating novel signaling pathways involved in erythropoiesis. IUBMB Life 2012; 64: 402-410.
[5] Palis J. Primitive and definitive erythropoiesis in mammals. Frontiers in physiology 2014; 5: 3.

Sample description

J2E model of Erythocytic differentiation

J2E cells are murine fetal liver cells that have been immortalised with the J2 retrovirus. J2E cells retain the capacity to respond to Epo by terminally differentiating and synthesizing hemoglobin6. The mouse J2E cell line responds to Epo by activating the JAK/STAT and ras/MAP kinase pathways7, as well as a novel Lyn-signaling cascade that we identified8. As a consequence of exposure to Epo, the cells undergo a burst of proliferation, followed by entry into the terminally differentiated state by synthesizing hemoglobin and changing morphologically9. These cells, therefore, provide a very good model for normal erythroid maturation in response to Epo.
J2E cells are maintained in DMEM (Gibco) 5% FCS (Bovogen Biologicals) at 370C and 5% CO2. Cell density is kept at 5-8 X105 cells/ml. Cells were induced with 5U/ml of Epo (Eprex®) (Jannsen). At least 1 X 107 cells were collected for RNA at 0min, 15min, 30min, 45min,1h, 1h 20min, 1h 40min, 2h, 2h 30min,3h, 3h 30min, 4h, 6h, 12h, 24h and 48h.

Key marker for differentiation
Enumeration of benzidine positive cells, as an indication of hemoglobin synthesis, was carried out to monitor differentiation. The time course of Epo-induced differentiation of J2E cells shows that hemoglobin production increases markedly 24-48h after stimulation (Figure 1).


Figure 1. Benzidine positive cells were enumerated at each time point. Three biological replicates were analysed.

Quality control

Expression of the following genes was assessed to determine the validity of this cell line as a model of Epo-induced erythroid differentiation (Figure 2). All these genes are required for normal erythroid differentiation.

  • Epor mediates epo-induced proliferation and differentiation [10]
  • Alas2 is the rate limiting enzyme for the Heme biosynthesis pathway [11]
  • Hbb-b1 hemoglobin, adult beta major chain required for oxygen transport [12]
  • Gata-1 is an essential transcription factor for erythroid development [13, 14]
  • Klf1 is a key transcriptional regulator for erythroid development [15]
  • Nfe2 regulates erythroid maturation [16]


Figure 2. Expression of key genes associated with erythroid differentiation. TPM: Tags per million.

Refereneces:
[6] Klinken SP, Nicola NA, Johnson GR. In vitro-derived leukemic erythroid cell lines induced by a raf- and myc-containing retrovirus differentiate in response to erythropoietin. Proc Natl Acad Sci U S A 1988; 85: 8506-8510.
[7] Tilbrook PA, Bittorf T, Callus BA, Busfield SJ, Ingley E, Klinken SP. Regulation of the erythropoietin receptor and involvement of JAK2 in differentiation of J2E erythroid cells. Cell Growth Differ 1996; 7: 511-520.
[8] Tilbrook PA, Ingley E, Williams JH, Hibbs ML, Klinken SP. Lyn tyrosine kinase is essential for erythropoietin-induced differentiation of J2E erythroid cells. EMBO J 1997; 16: 1610-1619.
[9] Busfield SJ, Klinken SP. Erythropoietin-induced stimulation of differentiation and proliferation in J2E cells is not mimicked by chemical induction. Blood 1992; 80: 412-419.
[10] Lodish HF, Hilton DJ, Klingmuller U, Watowich SS, Wu H. The erythropoietin receptor: biogenesis, dimerization, and intracellular signal transduction. Cold Spring Harb Symp Quant Biol 1995; 60: 93-104.
[11] Meguro K, Igarashi K, Yamamoto M, Fujita H, Sassa S. The role of the erythroid-specific delta-aminolevulinate synthase gene expression in erythroid heme synthesis. Blood 1995; 86: 940-948.
[12] Stamatoyannopoulos G. Control of globin gene expression during development and erythroid differentiation. Exp Hematol 2005; 33: 259-271.
[13] Tsai SF, Martin DI, Zon LI, D'Andrea AD, Wong GG, Orkin SH. Cloning of cDNA for the major DNA-binding protein of the erythroid lineage through expression in mammalian cells. Nature 1989; 339: 446-451.
[14] Whitelaw E, Tsai SF, Hogben P, Orkin SH. Regulated expression of globin chains and the erythroid transcription factor GATA-1 during erythropoiesis in the developing mouse. Mol Cell Biol 1990; 10: 6596-6606.
[15] Miller IJ, Bieker JJ. A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Kruppel family of nuclear proteins. Mol Cell Biol 1993; 13: 2776-2786.
[16] Andrews NC, Erdjument-Bromage H, Davidson MB, Tempst P, Orkin SH. Erythroid transcription factor NF-E2 is a haematopoietic-specific basic- leucine zipper protein. Nature 1993; 362: 722-728.

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


13063-139I3J2E erythroblastic leukemia response to erythropoietin00hr00minbiol_rep1
13064-139I4J2E erythroblastic leukemia response to erythropoietin00hr15minbiol_rep1
13065-139I5J2E erythroblastic leukemia response to erythropoietin00hr30minbiol_rep1
13066-139I6J2E erythroblastic leukemia response to erythropoietin00hr45minbiol_rep1
13067-139I7J2E erythroblastic leukemia response to erythropoietin01hr00minbiol_rep1
13068-139I8J2E erythroblastic leukemia response to erythropoietin01hr20minbiol_rep1
13069-139I9J2E erythroblastic leukemia response to erythropoietin01hr40minbiol_rep1
13070-140A1J2E erythroblastic leukemia response to erythropoietin02hr00minbiol_rep1
13071-140A2J2E erythroblastic leukemia response to erythropoietin02hr30minbiol_rep1
13072-140A3J2E erythroblastic leukemia response to erythropoietin03hr00minbiol_rep1
13073-140A4J2E erythroblastic leukemia response to erythropoietin03hr30minbiol_rep1
13074-140A5J2E erythroblastic leukemia response to erythropoietin04hrbiol_rep1
13075-140A6J2E erythroblastic leukemia response to erythropoietin06hrbiol_rep1
13076-140A7J2E erythroblastic leukemia response to erythropoietin12hrbiol_rep1
13077-140A8J2E erythroblastic leukemia response to erythropoietin24hrbiol_rep1
13078-140A9J2E erythroblastic leukemia response to erythropoietin48hrbiol_rep1
13129-140G6J2E erythroblastic leukemia response to erythropoietin00hr00minbiol_rep2
13130-140G7J2E erythroblastic leukemia response to erythropoietin00hr15minbiol_rep2
13132-140G9J2E erythroblastic leukemia response to erythropoietin00hr45minbiol_rep2
13133-140H1J2E erythroblastic leukemia response to erythropoietin01hr00minbiol_rep2
13134-140H2J2E erythroblastic leukemia response to erythropoietin01hr20minbiol_rep2
13135-140H3J2E erythroblastic leukemia response to erythropoietin01hr40minbiol_rep2
13136-140H4J2E erythroblastic leukemia response to erythropoietin02hr00minbiol_rep2
13137-140H5J2E erythroblastic leukemia response to erythropoietin02hr30minbiol_rep2
13138-140H6J2E erythroblastic leukemia response to erythropoietin03hr00minbiol_rep2
13139-140H7J2E erythroblastic leukemia response to erythropoietin03hr30minbiol_rep2
13140-140H8J2E erythroblastic leukemia response to erythropoietin04hrbiol_rep2
13141-140H9J2E erythroblastic leukemia response to erythropoietin06hrbiol_rep2
13142-140I1J2E erythroblastic leukemia response to erythropoietin12hrbiol_rep2
13143-140I2J2E erythroblastic leukemia response to erythropoietin24hrbiol_rep2
13144-140I3J2E erythroblastic leukemia response to erythropoietin48hrbiol_rep2
13195-141E9J2E erythroblastic leukemia response to erythropoietin00hr00minbiol rep3
13196-141F1J2E erythroblastic leukemia response to erythropoietin00hr15minbiol_rep3
13197-141F2J2E erythroblastic leukemia response to erythropoietin00hr30minbiol_rep3
13198-141F3J2E erythroblastic leukemia response to erythropoietin00hr45minbiol_rep3
13199-141F4J2E erythroblastic leukemia response to erythropoietin01hr00minbiol_rep3
13200-141F5J2E erythroblastic leukemia response to erythropoietin01hr20minbiol_rep3
13201-141F6J2E erythroblastic leukemia response to erythropoietin01hr40minbiol_rep3
13202-141F7J2E erythroblastic leukemia response to erythropoietin02hr00minbiol rep3
13203-141F8J2E erythroblastic leukemia response to erythropoietin02hr30minbiol_rep3
13204-141F9J2E erythroblastic leukemia response to erythropoietin03hr00minbiol_rep3
13205-141G1J2E erythroblastic leukemia response to erythropoietin03hr30minbiol_rep3
13206-141G2J2E erythroblastic leukemia response to erythropoietin04hrbiol_rep3
13207-141G3J2E erythroblastic leukemia response to erythropoietin06hrbiol_rep3
13208-141G4J2E erythroblastic leukemia response to erythropoietin12hrbiol_rep3
13209-141G5J2E erythroblastic leukemia response to erythropoietin24hrbiol_rep3
13210-141G6J2E erythroblastic leukemia response to erythropoietin48hrbiol_rep3
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