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

• We generated transgenic mice expressing fluorescent ubiquitination-based cell cycle indicator (Fucci) probes.
• Transgenic mice expressed Fucci probes at high levels in most hematopoietic cell populations.
• The fluorescence intensity of Fucci can be used for purification of hematopoietic stem cells.

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/G2/M and FucciG1, which label the nuclei of cells in S/G2/M phase green and those in G1 phase red, respectively. In this study, we generated Fucci transgenic mice and analyzed transgene expression in hematopoietic cells using flow cytometry. The FucciS/G2/M-#474 and FucciG1-#639 mouse lines exhibited high-level transgene expression in most hematopoietic cell populations. The FucciG1-#610 line expressed the transgene at high levels predominantly in the hematopoietic stem cell (HSC) population. Analysis of the HSC (CD34−KSL: CD34−/lowc-Kit+Sca-1+lineage marker−) population in the transgenic mice expressing both FucciS/G2/M and FucciG1 (#474/#610) confirmed that more than 95% of the cells were in G0/G1 phase, although the FucciG1(red) intensity was heterogeneous. An in vivo competitive repopulation assay revealed that repopulating activity resided largely in the FucciG1(red)high fraction of CD34−KSL cells. Thus, the CD34−KSL HSC population can be further purified on the basis of the Fucci intensity.