Reticulocytes. I. Isolation and in vitro maturation of synchronized populations

Studies of reticulocyte maturation have been limited by the inability to obtain pure populations of age-synchronized reticulocytes and by the absence of well-defined methods for the maturation of reticulocytes in vitro. Many of these problems were overcome using temporary suppression of erythropoiesis with thiamphenicol and phlebotomy resulting in a highly reproducible reticulocyte response, Percoll density gradient separation of cells yielding essentially pure populations of age-synchronized reticulocytes, and liquid culture techniques where cell lysis is minimal. The system allows reproducible study of well-defined cohorts of reticulocytes as they mature into erythrocytes. During in vitro maturation we serially monitored changes in reticulocyte count, glucose consumption, 125I-transferrin binding, fluorescein (FITC)-labeled transferrin binding, the activities of four erythrocyte enzymes (glucose-6-phosphate dehydrogenase, pyruvate kinase, phosphofructokinase, and lactate dehydrogenase) and the appearance of cells on scanning electron microscopy. These variables changed at different rates suggesting that multiple mechanisms underlie these maturational events. Transferrin binding and reticulocyte count decreased most rapidly and reached values near zero after three to four days in culture. The four enzyme activities decreased much more slowly, and only two reached pretreatment values after seven days in culture. In contrast to the findings of others, scanning electron microscopy suggested that cells do not assume the normal biconcave shape in this system. The methods described make it feasible to study the process of reticulocyte maturation in vitro. The data presented represent a first step in the study of the coordination and interrelationships of various maturational processes.