Dynamics of Membrane Protein Expression During Human Erythropoiesis: Resolving Distinct Maturational Stages of Erythroid cells

Abstract 811

Erythropoiesis is the process during which multipotential hematopoietic stem cells proliferate, differentiate and generate mature erythrocytes. Eight distinct developmental stages have been identified which include two functionally defined erythroid progenitor cells, the burst-forming unit-erythroid (BFU-E) and the colony-forming unit-erythroid (CFU-E). The other six morphologically distinguishable stages consist of proerythroblast, basophilic erythroblast, polychromatic erythroblast, orthochromatic erythroblast, reticulocyte and mature erythrocyte. During this differentiation process many dramatic cellular and membrane changes occur which include decrease in cell size, enhanced chromatin condensation, progressive hemoglobinization, enucleation and assembly of a unique erythrocyte membrane. We have recently demonstrated that during murine erythropoiesis, membrane proteins undergo ordered assembly. Of particular note, surface expression of CD44 exhibited a progressive and a dramatic 30-fold decrease from proerythroblast to reticulocyte. Importantly, use of surface expression of CD44 and glycophorin A in conjunction with cell size has enabled us to devise a new strategy for distinguishing unambiguously distinct stages of erythroid cells during terminal murine erythroid differentiation (Chen, et al PNAS, 2009). In the present study, we examined the expression of membrane proteins during human erythropoiesis by studying expression of various membrane proteins during in vitro induced erythroid differentiation of CD34+ hematopoietic stem cells from cord blood in two phase culture system. We found a number of similarities and some differences in the expression pattern of various membrane proteins between human and mouse erythroblasts. While surface expression of CD44 also decreased during terminal erythroid differentiation of human cells, the extent of decrease was half that seen for mouse cells. Furthermore, while expression of LU and ICAM-4 decreased during terminal differentiation of mouse cells, the expression of these two proteins involved in adhesive interactions increased in human cells. Surface expression of the anion channel, band 3 and glucose transporter, GLUT1 increased dramatically at the late stages of erythroid differentiation. Using CD44 and band 3 (or GLUT1) as surface markers, we were able to obtain pure populations of human erythroblasts (~90%) at distinct stages of erythroblasts from proerythroblasts to orthochromatic erythroblasts. Furthermore, we also examined the expression of CD34, IL3 receptor and CD36 during early stages of differentiation of pure populations of CD34+ cells during the first phase of the two phase culture system. While expression of CD34 and IL3 receptor are decreased with time, expression of CD36 increased over time. Colony forming assay revealed that while sorted CD34⁺CD36⁻IL3R⁺ cells were significantly enriched for BFU-e progenitors, CD34⁻CD36⁺IL-3R⁻ cells were predominantly CFU-e progenitors. These findings have thus enabled us to clearly distinguish erythroid progenitors and erythroblasts at successive developmental stages during human erythropoiesis and in conjunction with cell sorting obtain highly pure populations of erythroblasts at distinct stages of maturation. The experimental strategy we have developed should enable quantitative assessment of defining stage-specific defects in erythroid maturation in inherited and acquired red cell disorders and in bone marrow failure syndromes.