A primary human model system for iron restriction of erythropoiesis. (A) Effect of transferrin saturation (Sat) on growth and viability of hematopoietic lineages. CD34⁺ cells underwent unilineage culture in serum-free media. Results show mean ± SEM for 3 to 5 independent experiments. (B) Inhibition of early erythroid differentiation by iron restriction. Erythroid cultures with 100% or 15% transferrin saturation underwent flow cytometry (fluorescence-activated cell sorting [FACS]) with gating on viable cells. CD34/CD36 expression was analyzed on day 4. Indicated are percentages of cells in various quadrants. (C) Minimal apoptosis associated with erythroid iron restriction. Cells from panel B were analyzed by FACS for 7-amino-actinomycin D (7-AAD) and annexin V staining with gating on all cells. Whole cell lysates from day 5 cultures underwent immunoblotting (IB) for caspase 3 (bottom; arrow indicates cleavage product). (D) Minimal effect of erythroid iron restriction on cell-cycle parameters. Day 5 cultures stained with propidium iodide underwent FACS. Indicated are percentages of cells in various fractions, including subdiploid (Sub).