The erythrocyte is one of the best characterized human cells. However, studies of the process whereby human reticulocytes mature to erythrocytes have been hampered by the difficulty of obtaining sufficient numbers of cells for analysis. In the present study, we describe an in vitro culture system producing milliliter quantities of functional mature human adult reticulocytes from peripheral blood CD34 ؉ cells. We show that the final stage of reticulocyte maturation occurs by a previously undescribed mechanism in which large glycophorin A-containing vesicles forming at the cytosolic face of the plasma membrane are internalized and fuse with autophagosomes before expulsion of the autophagosomal contents by exocytosis.Early reticulocyte maturation is characterized by the selective elimination of unwanted plasma membrane proteins (CD71, CD98, and ␤1 integrin) through the endosome-exosome pathway. In contrast, late maturation is characterized by the generation of large glycophorin Adecorated vesicles of autophagic origin. IntroductionThe human erythrocyte is one of the best characterized mammalian cells, yet the process through which the enucleated erythroblast (reticulocyte) is converted to an erythrocyte remains poorly understood. Two distinct stages in reticulocyte maturation are evident from microscopic studies in animals. 1 Reticulocytes formed immediately after enucleation (denoted R1) are motile, multilobular, and normally confined to the BM. Mature (R2) reticulocytes are nonmotile and much more mechanically stable than their multilobular predecessors and are released from the BM into the peripheral circulation. 1,2 From the moment of enucleation until formation of the erythrocyte, the reticulocyte must lose approximately 20% of its surface area, reduce its volume, and degrade or eliminate residual cytosolic organelles. Current opinion is that the loss of surface area and degradation or elimination of residual organelles is achieved through 2 separate mechanisms. Plasma membrane loss is assumed to occur through the multivesicular endosome-exosome pathway in which small plasma membrane vesicles are endocytosed and incorporated into multivesicular endosomal bodies that subsequently fuse with the plasma membrane, releasing unwanted material as exosomes. 3,4 Degradation and elimination of organelles is effected by autophagy, a process whereby unwanted materials are enclosed in a double membrane to form autophagosomes that are delivered to lysosomes and expelled from the cell. 4 Substantial evidence suggests the endocytic and autophagic pathways converge. 5 In addition, fusion of multivesicular bodies (which are derived from endosomes) and autophagosomes has been described in the erythroleukemic cell line K562. 6 Early (R1) and late (R2) reticulocytes have different morphological and mechanical properties, but few studies have considered that different processes might be operative in the 2 cell types.A clearer understanding of this final step in the maturation of human erythroid cells would facilitate the study of ...

With increasing worldwide demand for safe blood, there is much interest in generating red blood cells in vitro as an alternative clinical product. However, available methods for in vitro generation of red cells from adult and cord blood progenitors do not yet provide a sustainable supply, and current systems using pluripotent stem cells as progenitors do not generate viable red cells. We have taken an alternative approach, immortalizing early adult erythroblasts generating a stable line, which provides a continuous supply of red cells. The immortalized cells differentiate efficiently into mature, functional reticulocytes that can be isolated by filtration. Extensive characterization has not revealed any differences between these reticulocytes and in vitro-cultured adult reticulocytes functionally or at the molecular level, and importantly no aberrant protein expression. We demonstrate a feasible approach to the manufacture of red cells for clinical use from in vitro culture.