The design and evaluation of therapies for the sickle cell and beta-thalassemia 
syndromes rely on our understanding of hemoglobin accumulation during human 
erythropoiesis. Here we report direct measurements of hemoglobin composition and 
messenger RNA (mRNA) levels in cultured CD34(+) cells and correlate those 
measurements with studies of freshly obtained bone marrow samples. Hemoglobin 
levels in differentiating cells were also compared with morphologic, 
immunophenotypic, and cell cycle assessments. A population of large 
preproerythroblasts was first identified within 24 hours and became the dominant 
population by day 5. The transition from proerythroblast to basophilic 
normoblast occurred later, from days 7 to 9, and correlated with a peak of 74.1% 
+/- 3.9% of the cells in the S phase of cell cycle. Orthochromatic normoblasts 
were the dominant and final cell type by day 13. High-performance liquid 
chromatography-based quantitation of fetal (HbF) and adult (HbA) hemoglobin and 
real-time polymerase chain reaction globin mRNA quantitation demonstrated a 
coordinate rise in the accumulation of both proteins and mRNA among these 
developmentally staged populations. Quantitative analyses on freshly sorted bone 
marrow populations demonstrated a similar rising pattern with beta-globin and 
HbA as the dominant species at both early and late stages of differentiation. We 
found no evidence for HbF dominant populations or switching during 
differentiation in adult cells. Instead, rapid increases in both HbF 
(heterocellular) and HbA (pancellular) content were observed, which coincided 
with the apex in cell cycling and the proerythroblast-basophilic normoblast 
transition. Based on these measurements, we conclude that HbF and HbA content 
are regulated with the rate of proliferation during adult erythropoiesis.