Figure 5.

In Vivo Counterparts of DP and CD41SP Cells

(A) Presence of CD41SP and DP cells in the embryo proper (EP) and yolk sac (YS) of E8.5 and E9.5 embryos. GFP-Tg males were crossed with non-Tg females to avoid possible contamination of maternal cells in the analysis. Note that GFP^low cell populations in the left shoulders of the gated CD45⁻TER119⁻EGFP⁺ cells were primitive erythrocytes.

(B) Myeloid lineage colonies developed from E9.5 CD41SP and DP cells on OP9 cells after culturing for 2 weeks. Scale bars, 200 μm.

(C and D) CD41SP cells isolated from E9.5 YS were cultured on OP9 cells for 5 days (C) or 3 weeks in the presence of interleukin-7 (D). Then, they were analyzed for the presence of myeloid cells (C) or B lymphocytes (D) by flow cytometry. A representative May-Grunwald/Giemsa-stained cytospin image is also shown in (C). m, macrophage; n, neutrophil; e, erythroblast. Scale bar, 50 μm.

(E) E9.5 CD41SP cells isolated from EP or YS cells were cultured for 1 day on OP9 and then analyzed for β-globin expression by RT-PCR to determine whether primitive erythrocytes were present. The expression of Hprt was evaluated as an internal control. Representative results from at least two separate experiments are shown.