Figure 1. Glucocorticoids sustain massive generation of proerythroblasts in culture by preventing their maturation in response to EPO.

(A) Fold Increase, flow cytometry analyses (forward/size side scatter and CD36/CD236a expression) and May-Grunwald Giemsa staining of cells obtained after 11 days in cultures of light density blood cells of normal adult volunteers stimulated either with multi-lineage GFs (SCF, IL-3, and EPO) or with GFs and DXM in combination (HEMA conditions), as indicated. Fold increase is calculated with respect to the number of cells seeded at day 0 and is presented as mean (±SD) of 10 separate experiments, each one with an individual donor. The difference in FI observed between cultures stimulated with GFs and those stimulated with GFs + DXM is statistically significant (<0.001) by paired t-test. By flow cytometry, cultures stimulated with GFs contain both erythroid (CD235a^pos) and non-erythroid (neutrophils, CD235a^dim, and lymphocytes, CD235a^neg) cells. By contrast, the majority (>90–95%) of the cells in cultures stimulated with GFs and DXM were erythroid (CD235a^pos). Furthermore, both by flow cytometry (lack of CD235a^highCD36^dim) and morphological criteria, erythroid cells obtained in the presence of GFs and DXM remained immature. Cell morphology is presented at 40× original magnification.

(B) May-Grunwald-Giemsa staining, forward size scatter (FSC) analyses and semiquantitative RT-PCR analyses for the expression of GATA1, GATA2 and Actin (the triangle on the top indicates amplifications levels after 25, 27 and 30 cycles), and quantitative RT-PCR analysis for β-globin in proerythroblasts obtained after 11 days of HEMA culture incubated for 24 h either under HEMA conditions (Prol) or in medium containing EPO alone. The levels of β-globin expression are presented as mean (±SD) of 5 separate experiments (*, p<0.01 by paired t test). Original magnification 40X.

(C) Flow cytometry analysis for Annexin V and propidium iodide staining of proerythroblasts generated in 11 days of HEMA cultures (GFs + DXM) and GF-starved for up to 6 h. Significant numbers (17%) of Annexin V^pos cells became detectable within 3 h of GF-starvation and ~25% of them became Annexin V and/or propidium iodide positive by 6 h.