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.