American Society of Hematology

Figure 7

$Figure 7. ESAM marks early hematopoietic progenitors throughout life. (A) Rag1/GFP− cells were sorted from AGM of E10.5 Rag1/GFP knockin heterozygous fetuses with high purity. The sorted Rag1/GFP− cells were incubated with the anti-ESAM Ab (1G8) followed by goat antirat IgG-FITC. The cells were then stained with anti-c-kit-APC, anti-Tie2-PE, and 7-AAD. The profile of Tie2 and c-kit expression in AGM cells was shown in a left panel. ESAM expression (solid line) in the Tie2Hi fraction of AGM and its control level with an isotype-matched IgG (dashed line) are presented (middle). The Tie2Hi ESAM+ cells (yellow) were c-kit+, whereas the Tie2Hi ESAM− cells (pink) were c-kit− (right). (B) The Tie2Hi ESAM− and Tie2Hi ESAM+ cells were sorted from E10.5 AGM and cultured on MS5 for 10 days. The recovered cells were counted and stained for the markers, including CD19 and Mac1. (C) Rag1/GFP− cells sorted from E10.5 YS were stained in the same manner as for the E10.5 AGM cells described in panel A. The profile of Tie2 and c-kit expression is shown in the top panel. In the lower panel, ESAM expression in the Tie2Hi c-kitLo fraction (tinted histogram) and the Tie2Lo c-kitHi fraction (open histogram) is shown. A dashed line shows the background fluorescence with an isotype-matched IgG. (D) ESAM expression of Rag1/GFP− Lin− ckitHi Sca1+ cells of adult bone marrow of 3-, 6-, 12-, and 18-month-old mice was analyzed. Representative flow cytometry results in 3 to 5 mice of each age were shown. ESAM expression on Rag1/GFP− Lin− c-kitHi Sca1+ cells of adult bone marrow of 3-, 12-, and 18-month-old mice (n = 3 in each) were simultaneously analyzed, and the data were summarized with respect to the mean fluorescence intensity with an anti-ESAM Ab or its control Ab (right panel). (E) ESAM−/Lo or ESAMHi cells were sorted from the Rag1/GFP− Lin− ckitHi Sca1+ fraction of the indicated adult bone marrow (7 and 20 months old, respectively) and subjected to methylcellulose colony assay. The data are from one of 2 independent experiments that gave similar results. Statistical significance: *P < .05, **P < .01. The percentages of cells in each gate are indicated in each panel.$

ESAM marks early hematopoietic progenitors throughout life. (A) Rag1/GFP⁻ cells were sorted from AGM of E10.5 Rag1/GFP knockin heterozygous fetuses with high purity. The sorted Rag1/GFP⁻ cells were incubated with the anti-ESAM Ab (1G8) followed by goat antirat IgG-FITC. The cells were then stained with anti-c-kit-APC, anti-Tie2-PE, and 7-AAD. The profile of Tie2 and c-kit expression in AGM cells was shown in a left panel. ESAM expression (solid line) in the Tie2^Hi fraction of AGM and its control level with an isotype-matched IgG (dashed line) are presented (middle). The Tie2^Hi ESAM⁺ cells (yellow) were c-kit⁺, whereas the Tie2^Hi ESAM⁻ cells (pink) were c-kit⁻ (right). (B) The Tie2^Hi ESAM⁻ and Tie2^Hi ESAM⁺ cells were sorted from E10.5 AGM and cultured on MS5 for 10 days. The recovered cells were counted and stained for the markers, including CD19 and Mac1. (C) Rag1/GFP⁻ cells sorted from E10.5 YS were stained in the same manner as for the E10.5 AGM cells described in panel A. The profile of Tie2 and c-kit expression is shown in the top panel. In the lower panel, ESAM expression in the Tie2^Hi c-kit^Lo fraction (tinted histogram) and the Tie2^Lo c-kit^Hi fraction (open histogram) is shown. A dashed line shows the background fluorescence with an isotype-matched IgG. (D) ESAM expression of Rag1/GFP⁻ Lin⁻ ckit^Hi Sca1⁺ cells of adult bone marrow of 3-, 6-, 12-, and 18-month-old mice was analyzed. Representative flow cytometry results in 3 to 5 mice of each age were shown. ESAM expression on Rag1/GFP⁻ Lin⁻ c-kit^Hi Sca1⁺ cells of adult bone marrow of 3-, 12-, and 18-month-old mice (n = 3 in each) were simultaneously analyzed, and the data were summarized with respect to the mean fluorescence intensity with an anti-ESAM Ab or its control Ab (right panel). (E) ESAM^−/Lo or ESAM^Hi cells were sorted from the Rag1/GFP⁻ Lin⁻ ckit^Hi Sca1⁺ fraction of the indicated adult bone marrow (7 and 20 months old, respectively) and subjected to methylcellulose colony assay. The data are from one of 2 independent experiments that gave similar results. Statistical significance: *P < .05, **P < .01. The percentages of cells in each gate are indicated in each panel.

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