title: 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/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(-/low)c-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.
authors.affiliation: Subteam for Manipulation of Cell Fate, BioResource Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan; Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Yo
authors.affiliation: Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Asako
authors.initials: A
authors.lastname: Sakaue-Sawano
authors.affiliation: Subteam for Manipulation of Cell Fate, BioResource Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Noda
authors.affiliation: Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
authors.affiliation: Subteam for Manipulation of Cell Fate, BioResource Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan. Electronic address: hiromiyoshi@keio.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Miyoshi
mesh_terms: D000818:Animals; D002453:Cell Cycle; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism; D008822:Mice, Transgenic; D019076:Transgenes; D054875:Ubiquitination
chemical_list: D005456:Fluorescent Dyes
keywords: Cell cycle; Cell cycle indicator; Hematopoietic cells; Hematopoietic stem cells; Transgenic mouse