Notch signaling maintains a CD45RA⁻cell subset in cultures of umbilical CB HSCs. (A) Flow cytometric analysis of lineage-depleted CD135⁺. Three populations of CD45RA were observed (CD45RA⁻, CD45RA^int, and CD45RA^hi). CD45RA⁻ were analyzed for CD133 expression and Rhodamine-1,2,3 (Rho) uptake which identified 3 levels of Rho staining (Rho^low, Rho^int, Rho^hi). The CD133⁺Rho^low cells were analyzed for the expression of CD90 and CD135. CD90^low are referred to as HSC, and CD90⁻ cells are referred to as MPP. A representative experiment is shown from at least 10 independent analyses. (B) Cocultures were initiated with CD90^low cells (HSCs) placed on OP9-Ctrl, -DL4^hi, and -DL4^low cells and analyzed by flow cytometry at days 9, 16, and 21 of culture for the expression of CD34 as indicated. (C) CD34⁺CD7⁻ gated cells from the indicated cocultures were analyzed at days 9, 16, and 21 for the expression of CD38 and CD45RA. Numbers within each plot indicate the percentage of cells in the indicated gates. A representative experiment is shown, from at least 10 independent cultures.