The developmental progression of stress erythroid progenitors in vitro and in vivo. (A) Unfractionated bone marrow cells were cultured in SEEM for 7 days (top), and the cells were switched into SEDM for 5 days (bottom). Representative flow cytometry analysis of population I and II cells using Kit⁺Sca1⁺ gated cells analyzed for CD71 and Ter119 (left) and CD34 and CD133 (right). (B) GFP⁺ CD34⁺CD133⁺KS cells were sorted by flow cytometry (left) and cocultured with GFP⁻ bone marrow cells in SEEM for 7 days (center) and then switched SEDM (right) for 5 days. At each step, GFP⁺ cells were gated on Kit⁺Sca1⁺ and analyzed for CD34 and CD133 expression. (C) Percentage of donor-derived (GFP⁺) cells in the spleen on the indicated days posttransplant (left). Total numbers of donor-derived CD34⁺CD133⁺KS and CD34⁻CD133⁻KS cells on the indicated days posttransplant (right). Numbers indicate mean ± standard error of the mean. (D) Analysis of the mRNA expression of GDF15, BMP4, Ihh, Shh, and Dhh in the spleen and of Epo in the kidney on the indicated days posttransplant. mRNA expression of the indicated genes was determined by qRT-PCR and expressed relative to the housekeeping gene Gapdh (2^ΔΔCt). (E) Unfractionated GFP⁺ bone marrow cells were transplanted in GFP⁻ recipient mice on the indicated days posttransplant, and spleen GFP⁺ cells were analyzed as indicated at the top of the figure.