In vitro, but not in vivo, expansion of HSCs is Notch dependent. (A) Freshly sorted HSCs were transduced with dnMaml-GFP or control YFP-only retroviral constructs, and sorted on day 6 posttransduction as GFP⁺ and YFP⁺ cells, cultured on OP9-Ctrl, -DL4^hi -DL4^low cells for 2 weeks. Cultures were analyzed by flow cytometry for the expression of the indicated markers as shown for total coculture. Cells gated as GFP⁺ or YFP⁺CD34⁺CD7⁻CD38⁻ were further analyzed for the expression of CD45RA (34⁺7⁻38⁻). Additionally, CD45RA⁻ gated cells were analyzed for the expression of CD90 (34⁺7⁻38⁻RA⁻). Numbers within each plot indicate the percentage of cells in the indicated gates or quadrants. Representative plots from at least 3 independent experiments are shown. (B) Cellular fold expansion of CD45RA⁻ cells (5-10 × 10³) is shown for the indicated coculture conditions at day 13 or 18 of culture relative to the start of coculture (ie, following the initial 7-day retroviral transduction period). Bar graphs show mean and SE of 4 independent experiments. (C) Freshly sorted HSCs were transduced with dnMaml-GFP or control YFP-only lentiviral constructs, and sorted on day 3 posttransduction as GFP⁺ or YFP⁺ CD34⁺CD7⁻CD38⁻CD90^low (CD90^low) cells. Equal numbers of transduced GFP⁺ or YFP⁺ CD90^low cells were coinjected into sublethally irradiated NSG neonatal mice, and after 10 weeks, cells from the BM were analyzed by flow cytometry for the indicated markers. (C) Top rows indicate the frequency of donor cells in BM (CD45⁺) and expression of GFP and YFP is shown for CD45⁺-gated cells. Bottom rows show GFP⁺ and YFP⁺ gated cells as indicated. Numbers within each plot indicate the percentage of cells in the indicated gates or quadrants. (D) Absolute numbers of donor-derived CD90^low cells and (E) CD45RA⁻ cells from the BM of host mice, as in panel C. A representative experiment is shown, from 3 reconstituted mice.