Fucci-guided purification of hematopoietic stem cells with high repopulating activity

Abstract Fluorescent ubiquitination-based cell cycle indicator (Fucci) technology utilizing the cell cycle-dependent proteolysis of ubiquitin oscillators enables visualization of cell cycle progression in living cells. The Fucci probe consists of two chimeric fluorescent proteins, FucciS/G 2 /M and FucciG 1 , which label the nuclei of cells in S/G 2 /M phase green and those in G 1 phase red, respectively. In this study, we generated Fucci transgenic mice and analyzed transgene expression in hematopoietic cells using flow cytometry. The FucciS/G 2 /M-#474 and FucciG 1 -#639 mouse lines exhibited high-level transgene expression in most hematopoietic cell populations. The FucciG 1 -#610 line expressed the transgene at high levels predominantly in the hematopoietic stem cell (HSC) population. Analysis of the HSC (CD34 − KSL: CD34 −/low c-Kit + Sca-1 + lineage marker − ) population in the transgenic mice expressing both FucciS/G 2 /M and FucciG 1 (#474/#610) confirmed that more than 95% of the cells were in G 0 /G 1 phase, although the FucciG 1 (red) intensity was heterogeneous. An in vivo competitive repopulation assay revealed that repopulating activity resided largely in the FucciG 1 (red) high fraction of CD34 − KSL cells. Thus, the CD34 − KSL HSC population can be further purified on the basis of the Fucci intensity.