Mutation of Gdf15 impairs expansion of stress erythroid progenitors in vitro. (A) Analysis of relative Gdf15 expression levels in unfractionated bone marrow (control), isolated erythroid progenitors (progenitors), and stromal cells (stroma) after 7 days of SEEM culture. One-way analysis of variance followed by Tukey pairwise comparison was performed. (B-E) Unfractionated WT or Gdf15^−/− bone marrow cells were cultured for 7 days in SEEM. Nonadherent SEPs were collected and stained with fluorescent anti-Kit, anti-Sca1, and anti-CD133 antibodies. (B) Flow gating strategy schematic. (C) Representative flow cytometry analysis. (D) Percentage (left) and total number (right) of Kit⁺Sca1⁺ cells. (E) Percentage (left) and total number (right) of CD34⁺CD133⁺Kit⁺Sca1⁺ cells, Data are shown as individual subject and the mean ± SEM. (F-H) Unfractionated WT or Gdf15^−/− bone marrow cells were labeled with PKH26 dye at the beginning of a 5-day culture in SEEM. (F) Schematic of the gating strategy. (G) Representative flow cytometry analysis. (H) Percentage of PKH26^loCD133^negKit⁺Sca1⁺ cells (top) and PKH26^hiCD133⁺Kit⁺Sca1⁺ cells (bottom). Data are shown as individual subject and the mean ± SEM. *P < .05, **P < .01, and ***P < .001. ns, not significant.