CD34 and CD133 expression defines a series of immature stress erythroid progenitors. (A) Representative flow cytometry analysis of unfractionated mouse bone marrow prior to culture (left), after 7 days in SEEM (center), and after 5 days in SEEM + Epo or SEDM (right). Cells were gated on Kit⁺Sca1⁺, and the expression of CD34 and CD133 is shown. (B) Unfractionated mouse bone marrow was grown in SEEM for 7 days and switched into SEEM + Epo media. Representative flow cytometry analysis is shown for cells grown for 3 days and 7 days. (C) CD34⁺CD133⁺KS, CD34⁻CD133⁺KS, and CD34⁻CD133⁻KS cells were sorted from cultures by flow cytometry. A total of 2 × 10⁴ cells were plated in methylcellulose media containing Epo only to assay for mature stress BFU-Es or in media containing Epo + BMP4 + SCF at 2% O₂ to assay maximal stress BFU-E potential. (D) CD34⁺CD133⁺KS, CD34⁻CD133⁺KS, and CD34⁻CD133⁻KS cells were sorted from cultures by flow cytometry. mRNA expression of the indicated genes was determined by qRT-PCR and expressed relative to the housekeeping gene Gapdh (2^ΔΔCt).