2014 – FLUORESCENT CELL CYCLE TIMER ENABLED ANALYSIS OF NORMAL AND INEFFECTIVE ERYTHROPOIESIS

Abstract

Aberrant cell cycle speed during hematopoiesis defines pathologic conditions, such as the inability to compensate for anemia in diseases of ineffective erythropoiesis like hemolysis or thalassemia. We utilized a unique live-cell reporter of cell cycle speed using a Histone H2B-FT fusion protein containing the color-changing Fluorescent Timer (FT) protein, distinguishing faster cycling cells from slower-cycling ones based on the intracellular ratio between blue and red fluorescence. In transgenic mice expressing this reporter, we characterized the stress erythropoietic response of spleen and bone marrow following phenylhydrazine-induced hemolytic anemia. Using flow cytometric sorting of erythroblast stages, we found that all stages divide more rapidly after the hemolytic insult, not only early progenitors as previously thought. We also observe that stress erythropoiesis in the spleen is stimulated almost immediately after hemolysis. And the last nucleated cell stage, orthochromatophilic erythroblasts, stop dividing much earlier than normal, allowing them to terminally differentiate into reticulocytes much faster to alleviate anemia. Mass spectrometry of sorted populations shows upregulation of cell cycle and metabolic pathway related genes. Using this system in in vitro human CD34+ primary cell culture, we assessed the erythropoietic response to known treatments of β-thalassemia and Diamond Blackfan Anemia, like Epo and dexamethasone. We found an increase in cell cycling rates with an increase in dexamethasone concentration but not that of Epo. These findings shed new light on the normal response to external stress, underscoring the possibility of precise quantification of cell cycle speed in animal models of anemia. This system can help in understanding stress hematopoiesis, allowing identification of cellular factors that facilitate response to external stress.