American Society of Hematology

Figure 4

$Figure 4. The Eng promoter in combination with the −8 endothelial enhancer targets HECs enriched for hematopoietic potential. (A) Schematic diagram outlining the experimental procedure. Recombinant ES cells generated using the Eng reporter constructs were differentiated into day 3 EBs. FLK1+ mesodermal cells were sorted from representative clones for each recombinant ES cell line and cultured in LBM. At 48 hours, CD41−/TIE2+/c-KIT+ (HE) cells were sorted into lacZ+ and lacZ– fractions. The sorted fractions were recultured in LBM for a further 48 hours followed by flow cytometry and CFU-C assays. (B) (i) CD41 and TIE2 expression in sorted c-KIT+ HE LacZ− (white) and LacZ+ (blue) fractions (after 2 days of reculture) derived from Eng −8/P/LacZ ES cells. (ii) Percentage of CD45+ cells generated from LacZ− and LacZ+ HE fractions. (iii) Bar chart shows the number and type of hematopoietic colonies generated by each fraction. (C) (i-iii) Corresponding data to (B) generated from Eng −8/P/LacZ/+7/+9 ES cells. (D) (i-iii) Corresponding data to (B) generated from Eng −8/P/LacZ/+7 ES cells. (E) (i-iii) Corresponding data to (B) generated from Eng −8/P/LacZ/+9 ES cells. Primitive and definitive colonies were scored after 4 and 9 days, respectively. Statistical analysis was conducted using Student t test, *P < .05, **P < .01.$

The Eng promoter in combination with the −8 endothelial enhancer targets HECs enriched for hematopoietic potential. (A) Schematic diagram outlining the experimental procedure. Recombinant ES cells generated using the Eng reporter constructs were differentiated into day 3 EBs. FLK1+ mesodermal cells were sorted from representative clones for each recombinant ES cell line and cultured in LBM. At 48 hours, CD41−/TIE2+/c-KIT+ (HE) cells were sorted into lacZ+ and lacZ– fractions. The sorted fractions were recultured in LBM for a further 48 hours followed by flow cytometry and CFU-C assays. (B) (i) CD41 and TIE2 expression in sorted c-KIT+ HE LacZ− (white) and LacZ+ (blue) fractions (after 2 days of reculture) derived from Eng −8/P/LacZ ES cells. (ii) Percentage of CD45+ cells generated from LacZ− and LacZ+ HE fractions. (iii) Bar chart shows the number and type of hematopoietic colonies generated by each fraction. (C) (i-iii) Corresponding data to (B) generated from Eng −8/P/LacZ/+7/+9 ES cells. (D) (i-iii) Corresponding data to (B) generated from Eng −8/P/LacZ/+7 ES cells. (E) (i-iii) Corresponding data to (B) generated from Eng −8/P/LacZ/+9 ES cells. Primitive and definitive colonies were scored after 4 and 9 days, respectively. Statistical analysis was conducted using Student t test, *P < .05, **P < .01.

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