Differentiation Of Human Erythroid Progenitors Research Articles

Dynamic Changes in Membrane Protein Expression During Murine and Human Erythropoiesis: Resolving the Distinct Stages in Terminal Erythroid Differentiation.

Abstract 4039Poster Board III-975Erythropoiesis is the process by which nucleated erythroid progenitors proliferate and differentiate to generate, every second, millions of non-nucleated red cells with their unique discoid shape and membrane material properties. In the present study, we examined the time-course of appearance of individual membrane proteins during erythropoiesis to enhance our understanding of the evolution of the unique features of the red cell membrane. We found that all the major transmembrane and all the skeletal proteins in mature red blood cells, except actin, accrued progressively during terminal differentiation of murine erythroid progenitors. In marked contrast, during the same time course, accumulation of various adhesion molecules decreased. In particular, the adhesion molecule, CD44 exhibited a progressive and a dramatic 30-fold decrease from proerythroblast to reticulocyte; this enabled us to devise a new strategy for distinguishing unambiguously between erythroblasts at successive developmental stages during murine erythopoiesis. By contrast, CD71, which is routinely being used as a surface marker for this purpose, proved markedly less effective than CD44 in identifying erythroblasts at successive developmental stages. Indeed, we found that CD71 expression decreased only fourfold during terminal erythroid differentiation, and not in the progressive manner observed for CD44. A similar pattern of membrane protein expression was noted during terminal differentiation of human erythroid progenitors. These findings provide new insights into the genesis of red cell membrane function during erythroblast differentiation, and also offer a means of defining stage-specific defects in erythroid maturation in inherited and acquired red cell disorders and in bone marrow failure syndromes. Disclosures:No relevant conflicts of interest to declare.

Tyrosine Residues of the Erythropoietin Receptor Are Dispensable for Erythroid Differentiation of Human CD34+ Progenitors

To study the role of the cytoplasmic domain and particularly the tyrosine residues of the erythropoietin receptor (EpoR) in erythroid differentiation of human primary stem cells, we infected cord blood-derived CD34+ cells with retroviruses encoding chimeric receptors containing the extracellular domain of the prolactin receptor (PRLR) and the cytoplasmic domain of either the normal EpoR or a truncated EpoR devoid of tyrosine residues. Erythroid differentiation of the infected progenitors could thus be studied after stimulation by PRL. The complete PRLR was used to assess its ability to substitute for EpoR in erythroid differentiation. Typical erythroid day-14 colonies were observed from CD34+ cells grown in PRL when infected with any of the three viral constructs. These results demonstrate that: (i) the activation of the virally transduced PRLR leads to erythroid colony formation showing that erythroid terminal differentiation can be induced by a non-erythroid receptor in human progenitors; (ii) a chimeric receptor PRLR/EpoR is able to transduce a signal leading to terminal erythroid differentiation of human CD34+ cells; (iii) in contrast to results previously reported in murine models, tyrosine residues of the EpoR are not required for growth and terminal differentiation of human erythroid progenitors.

Cell surface antigen expression in human erythroid progenitors: erythroid and megakaryocytic markers.

This review summarizes the changes in cell surface antigen expression during proliferation and differentiation of human erythroid progenitors. The content is based on our experimental data obtained from complement-mediated cytotoxicity assays against hematopoietic progenitors and a combined technique of sequential micromanipulations of paired daughter cells derived from erythroid burst-forming units (BFU-E) and immunostaining with a panel of monoclonal antibodies, as well as from current information. BFU-E has CD34, CD41a (platelet glycoprotein[GP]IIb/IIIa) and CD41b(GPIIb) antigens. Paired daughter cells derived from BFU-E have CD41a, CD41b, CD71 (transferrin receptor) and HLA-DR antigens, but not CD34 or CD33 antigen. The CD36 antigen (thrombospondin receptor or GPIV) is first expressed on the cells after 5 days of culture, in agreement with the report that the anti-CD36 positive fraction contained a greater part of the erythroid colony-forming units (CFU-E). The blood group A antigen is first expressed on cells from aggregates derived from BFU-E after 5 days of culture. Glycophorin A is expressed on cell surface after 7 days of culture when proerythroblasts first appear. Hemoglobin alpha is expressed after 8 days of culture and coincides with the first appearance of basophilic erythroblasts. This review provides useful information on the identification of leukemic cells from poorly differentiated acute leukemias such as early erythroblastic leukemia and acute megakaryoblastic leukemia, and is useful in the understanding of the commitment and differentiation of erythroid and megakaryocytic progenitors in normal hematopoiesis.

Changes in cell surface antigen expressions during proliferation and differentiation of human erythroid progenitors

Cell surface antigen expression during proliferation and differentiation of human erythroid progenitors was examined using a combination of sequential micromanipulations of paired daughter cells derived from erythroid burst-forming units (BFU-E) and immuno-staining with a panel of monoclonal antibodies. Single hematopoietic progenitors were identified in methylcellulose cultures containing human cord blood mononuclear cells and micromanipulated individually to secondary culture. Paired daughter cells, granddaughter cells, and subsequent generations, whose counterparts produced erythroid bursts, were stained with various cytochemical and immuno-alkaline phosphatase stainings. Most paired daughter cells of BFU-E immunostained positively with anti-platelet glycoprotein(GP) IIb, antiplatelet GPIIb/IIIa, anti-HLA-DR, and antitransferrin receptor antibodies. Acid phosphatase staining was also positive. Neither CD34 nor CD33 antigens were identified on the cells. CD36 and blood group A antigens were first identified on cells from aggregates containing 32 to 64 cells after 4 days of secondary culture and preceded the expression of glycophorin A and hemoglobin alpha. These results indicate that various cell surface antigens were sequentially expressed during the proliferation and differentiation of erythroid progenitors, and that our procedure may be useful for clarifying the morphologic and immunologic properties of hematopoietic stem cells.

Changes in Cell Surface Antigen Expressions During Proliferation and Differentiation of Human Erythroid Progenitors

Abstract Cell surface antigen expression during proliferation and differentiation of human erythroid progenitors was examined using a combination of sequential micromanipulations of paired daughter cells derived from erythroid burst-forming units (BFU-E) and immuno-staining with a panel of monoclonal antibodies. Single hematopoietic progenitors were identified in methylcellulose cultures containing human cord blood mononuclear cells and micromanipulated individually to secondary culture. Paired daughter cells, granddaughter cells, and subsequent generations, whose counterparts produced erythroid bursts, were stained with various cytochemical and immuno-alkaline phosphatase stainings. Most paired daughter cells of BFU-E immunostained positively with anti- platelet glycoprotein(GP) IIb, antiplatelet GPIIb/IIIa, anti-HLA-DR, and antitransferrin receptor antibodies. Acid phosphatase staining was also positive. Neither CD34 nor CD33 antigens were identified on the cells. CD36 and blood group A antigens were first identified on cells from aggregates containing 32 to 64 cells after 4 days of secondary culture and preceded the expression of glycophorin A and hemoglobin alpha. These results indicate that various cell surface antigens were sequentially expressed during the proliferation and differentiation of erythroid progenitors, and that our procedure may be useful for clarifying the morphologic and immunologic properties of hematopoietic stem cells.

Growth of human normal erythroid progenitors in liquid culture: A comparison with colony growth in semisolid culture

Most studies of erythropoiesis in vitro have employed cloning methods in semisolid medium. We have recently described a two-step liquid culture procedure that supports the proliferation and differentiation of human erythroid progenitors. In the present study, we have modified the procedure to allow large-scale cultures of erythroid cells derived from normal donors. The culture is divided into two phases. In the first phase, which is erythropoietin (Epo) independent, the early erythroid progenitors multiply and differentiate. In the second, Epo-dependent phase, they mature into orthochromatic normoblasts and enucleated erythrocytes. Using this procedure, erythroid cell yield reached 7.5 x 10(6)/ml and a total of 7 x 10(8) cells could be harvested per blood unit. A comparison of the growth of erythroid cells in liquid culture to their colony growth in semisolid culture indicated that cell growth was superior: 1) in liquid culture in terms of cell yield per originally cultured mononuclear cell, 2) per ml culture and per culture surface area and in the purity of the resultant erythroid cell population. In addition, it permits easier manipulation of the culture condition and components and sampling of greater than 1 x 10(7) cells at each maturation stage subsequent to the proerythroblast stage. This liquid culture procedure might provide an important experimental tool for studying erythroid cell development.