In Vitro Murine Hematopoiesis Supported by Signaling from a Splenic Stromal Cell Line

Figure 5

The specific effect of Notch inhibition on hematopoiesis in vitro. LT-HSC (Lin⁻Sca-1⁺c-Kit⁺Flt3⁻CD150⁺) and MPP (Lin⁻Sca-1⁺c-Kit⁺Flt3⁺CD150⁻) were sorted from murine bone marrow and cultured above 5G3 stroma for a period of 21 days. Cocultures were supplemented twice weekly with DAPT (10 μM in DMSO), a γ-secretase inhibitor. Control cultures received DMSO diluent alone or NIL. Nonadherent cells were collected after 21 days and stained with antibodies specific for CD11b, CD11c, MHC-II, and F4/80 to delineate subsets. (a) Cells were delineated through marker expression as either L-DC (CD11b⁺CD11c^+/−MHC-II⁻F4/80⁺) or “progenitors” (CD11b⁻CD11c⁻MHC-II⁻F4/80⁻). Staining data of one representative experiment shows the production of predominantly L-DC. (b) The proportional representation of L-DC and the absolute number of L-DC produced for three replicate experiments involving separate cell sorts are shown. Data reflect the mean ± SE (). identifies a significant difference () in cell production in DAPT treated cocultures (10 μM) compared with NIL controls, except for % of L-DC which is significantly different from both NIL and diluent controls.

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