Novel Fluorescent Timer Tool Enables Characterization of Erythropoietic Differentiation Based on Differential Cell Cycling Speeds

Erythropoietic proliferation and differentiation are coordinated and regulated by a complex compendium of molecular components and networks. Understanding the underlying mechanisms and the dependence of erythroid maturation on cell-cycle behavior can provide a detailed insight into normal and ineffective erythropoiesis. The dynamic cell cycle speed of erythroid progenitors reflects the erythron’s response to external stimuli, such as severe anemia or bleeding. Aberrant cell cycle speed also defines pathologic conditions, such as the inability to compensate for anemia in diseases of ineffective erythropoiesis like hemolysis or thalassemia. Current methods to resolve cell cycle length heterogeneity at a single-cell level in real-time present with limitations, including cellular toxicity, insufficient intensity, and dilution over subsequent cell divisions. 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. The FT protein emits blue fluorescence when newly synthesized and matures into a stable red fluorescent protein over 1.2 hours. The fusion protein thus distinguishes faster cycling cells from slower-cycling ones based on the intracellular ratio between blue and red fluorescence.

Knock-in mice expressing H2B-FT from a universally active locus under the control of a dox inducible promoter were previously generated and characterized. We successfully characterized the stress erythropoietic response of the spleen and bone marrow (BM) after inducing hemolytic anemia by phenylhydrazine (PHZ) administration in these transgenic mice. Flow cytometric investigation of successive stages of erythroblasts revealed that all stages of erythroblasts maintain rapid cell division after the hemolytic insult (****p<0.0001, Mann-Whitney test) and not only early progenitors, as previously thought. We also observed that stress erythropoiesis in the spleen is stimulated almost immediately after hemolysis. Most importantly, we observed that the last nucleated cell stage, orthochromatic erythroblasts, stop dividing much earlier than normal, allowing them to terminally differentiate into reticulocytes much faster to alleviate the anemia.

Blue-red (BR) profiles of the different erythroblasts from the PHZ-treated animals showed a marked distribution into fast-cycling (high blue fluorescence) and slow-cycling (high red fluorescence) subpopulations. Histograms of normalized BR ratios revealed significantly differentially cycling subpopulations in the polychromatic erythroblasts from spleen and orthochromatic erythroblasts from BM under stress. Mass spectrometric analysis of the differentially cycling subpopulations sorted from the respective erythroblasts shows upregulation of genes encoding cell cycle related and phospho-proteins. We are currently performing comparative analyses with openly available proteomic data.

With the Erythropoietin (Epo) model for inducing stress erythropoiesis, we do find a modest increase in blue-red ratios for each of the erythroblast populations in Epo-treated timer mice as compared to the PHZ model.

A recent study on steroid resistance in DBA reported that dexamethasone (dex) treatment of peripheral blood progenitors caused the specific upregulation of p57Kip2 leading to higher expansion and accelerated erythroid differentiation. We will utilize in vitro human CD34+ primary cell culture to assess the erythropoietic response to known treatments of anemia of chronic kidney disease and Diamond-Blackfan Anemia, like Epo and dex, respectively.

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. We highlight the use of a sophisticated fluorescent system that can help elucidate the role of cell cycle speed in stress hematopoiesis, and determine the mechanistic pathways acting at single-cell or population level. Further phosphoproteomic investigation can lead to identification of discrete molecular targets regulating erythroid cell proliferation and differentiation with potential therapeutic implications. The tool can aid in answering important questions delineating cell cycle dynamics as the cause or consequence of erythroid differentiation in normal and pathophysiological conditions.