Culture of unfractionated bone marrow cells leads to the development of stress BFU-Es. (A) Unfractionated bone marrow cells were cultured for the indicated number of days in IMDM supplemented with BMP4 + Shh + GDF15 + SCF + Epo and cultured at 2% O2. On the indicated days, 1 × 105 cells were plated in methylcellulose media containing BMP4 + SCF + Epo and cultured at 2% O2 for 5 days. Stress BFU-Es were scored after benzidine staining. (B) Flow cytometry analysis of unfractionated bone marrow cultures on day 6 of culture. Cells were gated on Kit+Sca1+ cells, and the percentages of CD71 and Ter119 are shown. (C) Unfractionated bone marrow cells were grown for 5 days in IMDM supplemented with BMP4 + Shh + GDF15 + SCF + Epo and cultured at 2% O2 (control media, C) or in control media with the indicated factor removed. A total of 5 × 104 cells were then plated in methylcellulose media supplemented with Epo, and stress BFU-E colonies were scored after 5 days. Data are representative of at least 2 independent experiments.
