American Society of Hematology

Figure 3

$Figure 3. Eng+Flk-1+ cells are endowed with hematopoietic and endothelial potential. (A) Flow cytometric analyses of WT E7.5 embryos for Eng (x-axis) and Flk-1 (y-axis). Gates represent Eng−Flk+ (E−F+), Eng+Flk+ (E+F+), and Eng+Flk− (E+F−) fractions that were sorted for further in vitro characterization (B-D). Numbers represent the percentage of gated cells that express a given surface antigen. (B) E−F+ (white bars), E+F+ (gray bars), and E+F− (black bars) cell fractions gated in panel A were further analyzed for the expression of hematopoietic and endothelial markers. Histograms represent the mean and error bars represent the SEM from at least 2 independent experiments. (C) Transcriptional analyses of E7.5 embryos based on Eng and Flk-1 expression. Clustering of genes found to be distinctively expressed among the E−F−, E−F+, E+F+, and E+F− fractions. Colors show the range of expression from blue (low expression) to red (high expression). Genes associated with endothelial, hematopoietic, and more primitive precursors were found to be highly expressed in the E+F+ fraction. (D) Frequency of hematopoietic progenitors in E−F−, E−F+, E+F+, and E+F− cells sorted from E7.5 embryos (representative gates are shown in panel A). Error bars indicate the SEM from 3 independent experiments. (E) Hematopoietic (round cells, left panel) and endothelial cells (arrows, left panel) emerged after the coculture of E+F+ cells onto OP9 stromal cells in the presence of hematopoietic and endothelial cytokines (10× objective). Staining for CD31 (green, right panel) confirmed the endothelial nature of these elongated cells. DAPI identifies nuclei (blue). Confocal micrograph represents results from 3 independent experiments (Zeiss LM510; 10× objective). (F) FACS characterization of cells showed in panel E after a 7-day in vitro expansion confirm the presence of hematopoietic (c-Kit+CD41+ and CD45+VE-Cad−) and endothelial (VE-Cad+Flk-1+ and VE-Cad+CD45−) cells in these cultures. Representative profiles are shown for c-Kit and CD41 (left panel), VE-Cad and Flk-1 (middle panel), and VE-Cad and CD45 (right panel).$

Eng⁺Flk-1⁺ cells are endowed with hematopoietic and endothelial potential. (A) Flow cytometric analyses of WT E7.5 embryos for Eng (x-axis) and Flk-1 (y-axis). Gates represent Eng⁻Flk⁺ (E⁻F⁺), Eng⁺Flk⁺ (E⁺F⁺), and Eng⁺Flk⁻ (E⁺F⁻) fractions that were sorted for further in vitro characterization (B-D). Numbers represent the percentage of gated cells that express a given surface antigen. (B) E⁻F⁺ (white bars), E⁺F⁺ (gray bars), and E⁺F⁻ (black bars) cell fractions gated in panel A were further analyzed for the expression of hematopoietic and endothelial markers. Histograms represent the mean and error bars represent the SEM from at least 2 independent experiments. (C) Transcriptional analyses of E7.5 embryos based on Eng and Flk-1 expression. Clustering of genes found to be distinctively expressed among the E⁻F⁻, E⁻F⁺, E⁺F⁺, and E⁺F⁻ fractions. Colors show the range of expression from blue (low expression) to red (high expression). Genes associated with endothelial, hematopoietic, and more primitive precursors were found to be highly expressed in the E⁺F⁺ fraction. (D) Frequency of hematopoietic progenitors in E⁻F⁻, E⁻F⁺, E⁺F⁺, and E⁺F⁻ cells sorted from E7.5 embryos (representative gates are shown in panel A). Error bars indicate the SEM from 3 independent experiments. (E) Hematopoietic (round cells, left panel) and endothelial cells (arrows, left panel) emerged after the coculture of E⁺F⁺ cells onto OP9 stromal cells in the presence of hematopoietic and endothelial cytokines (10× objective). Staining for CD31 (green, right panel) confirmed the endothelial nature of these elongated cells. DAPI identifies nuclei (blue). Confocal micrograph represents results from 3 independent experiments (Zeiss LM510; 10× objective). (F) FACS characterization of cells showed in panel E after a 7-day in vitro expansion confirm the presence of hematopoietic (c-Kit⁺CD41⁺ and CD45⁺VE-Cad⁻) and endothelial (VE-Cad⁺Flk-1⁺ and VE-Cad⁺CD45⁻) cells in these cultures. Representative profiles are shown for c-Kit and CD41 (left panel), VE-Cad and Flk-1 (middle panel), and VE-Cad and CD45 (right panel).

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