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First published online June 22, 2006
doi: 10.1242/10.1242/dev.02440

Development 133, 2771-2779 (2006)
Published by The Company of Biologists 2006
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Discordant developmental waves of angioblasts and hemangioblasts in the early gastrulating mouse embryo

Chie Furuta1, Hideo Ema1,*, Shin-ichiro Takayanagi1, Takunori Ogaeri1, Daiji Okamura2, Yasuhisa Matsui2 and Hiromitsu Nakauchi1,*

1 Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan.
2 Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University, 4-1 Seiryo-machi, Sendai, 980-8575, Japan.


Figure 1
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  		Fig. 1. Frequency of endothelial or hematopoietic progenitors in E7.5 embryos. (A) Stroma-dependent hematopoietic colonies (a,b) and vascular endothelial colonies (c,d) are representatively shown. These colonies were formed by cells obtained from E7.5 embryos in co-culture with OP-9 stromal cells. Endothelial colonies were immunostained with anti-CD31 antibody. Red, CD31; blue, DAPI. Scale bar: 100 µm. (B) Limiting-dilution analysis of endothelial progenitors and stroma-dependent hematopoietic progenitors in E7.5 embryos. Cells were plated in 96-well plates and were co-cultured with OP-9 stromal cells for 7 days in the presence of IL3, TPO, SCF, EPO and VEGF. The frequency of endothelial progenitors was estimated to be one in 0.05 embryo equivalents. The 95% confidence interval (CI) was 1/38-1/56. The frequency of stroma-dependent hematopoietic progenitors was estimated to be one in 0.14 embryo equivalent, and the 95% CI was 1/115-1/181.

 

Figure 2
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  		Fig. 2. Temporal kinetics of endothelial and hematopoietic progenitors in early gastrulating embryos. The numbers of endothelial and stroma-dependent hematopoietic progenitors, and CFU-c per embryo were measured in precisely staged embryos from E5.5 to E7.75. Five to 14 embryos were examined for endothelial and hematopoietic progenitors at each time point. Seven to 11 embryos were examined for CFU-c at each time point. Data are expressed in mean±s.d. per embryo. Sigmoid dose-response curves were used for approximation.

 

Figure 3
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  		Fig. 3. Replating of embryonic cells into methylcellulose. (A) E7.5 cells per embryo were co-cultured with OP-9 cells in the presence of IL3, TPO, SCF, EPO and VEGF. On day 0, 1, 2 or 4, after treatment with trypsin, single cells were replated into methylcellulose with IL3, TPO, SCF, EPO and VEGF. Following 7 days of further culture, colonies formed were picked up. Cytospin preparations were made and were stained with Hemacolor. This experiment used a total of 20 E7.5 embryos (three MS-, four LS-, seven OB-, three EB-, three LB-stage embryos). (B) Data from a total of four embryos are shown. Two embryos were of ES stage and two embryos were of MS stage. nmE, neutrophil/macrophage/erythrocyte; mE, macrophage/erythrocyte; m, macrophage. (C) RT-PCR analysis for ßH1 and ß-major globin expression in individual hematopoietic colonies that were formed after contact with OP-9 cells for 3 days.

 

Figure 4
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  		Fig. 4. Expression of Flk1, Tie2, CD31 or Oct3/4-GFP in E7.5 embryos. Representative flow cytometric analyses of pooled embryonic cells around E7.5 are shown. (A) Cells stained with an anti-Flk1 antibody. 6.0±11.1% (mean±s.d., n=9) of the cells showed expression of Flk1. (B) Cells stained with an anti-Tie2 antibody. 3.0±2.7% (mean±s.d., n=4) of the cells showed expression of Tie2. (C) Cells stained with an anti-CD31 antibody. 4.3±3.8% (mean±s.d., n=5) of the cells showed expression of CD31. (D) 39.0±25.3% (mean±s.d., n=6) of the cells from Oct3/4-GFP+/- embryos expressed GFP.

 

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