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

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(+)line-age 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. (C) 2014 Elsevier Inc. All rights reserved.