Figure 5. E4ORF1⁺ ECs prevent attrition of KLS cells through Notch activation.

A) Quantitative RT-PCR for assessing Notch 1 and Notch 2 expression on day 21 co-cultured hematopoietic cells. B) E4ORF1⁺ ECs were infected with adenovirus that overexpress a Notch decoy construct or an empty adenovirus. The Notch decoy significantly decreased the ability of E4ORF1⁺ ECs to expand total hematopoietic cells and total TNR.Gfp⁺KLS cells. C-F) Notch1^-/-Notch2^-/- lineage⁻ (N1N2^-/-) BM cells (25,000 cells) were co-cultured with E4ORF1⁺ ECs. BM cells were analyzed on weekly time points and were demi-depopulated each week. C) Total hematopoietic cell expansion was significantly decreased by day 21 in co-culture with Notch1^-/-Notch2^-/- versus WT BM cells. D) Total lineage⁻ hematopoietic cell expansion was significantly decreased by day 21 in co-culture of Notch1^-/- Notch2^-/- versus WT BM cells with E4ORF1⁺ ECs. E) Total lineage⁺ hematopoietic cell expansion was significantly decreased by day 21 in co-culture of Notch1^-/-Notch2^-/- BM cells versus WT cells E4ORF1⁺ ECs. F) Total CD34^-Flt3^-KLS cells (containing a large number of phenotypically marked HSCs) were significantly decreased by day 14 in co-cultures of E4ORF1⁺ ECs with Notch1^-/-Notch2^-/-versus WT BM cells. G) Representative contour plots of phenotypic analysis of Notch1^-/-Notch2^-/-CD34⁻ Flt3⁻KLS versus WT BM CD34⁻Flt3⁻KLS cells co-cultured for 7, 14, and 21 days with E4ORF1⁺ ECs. (*p<0.05, n = 6).