title: Direct observation of cell cycle progression in living mouse embryonic stem cells on an extracellular matrix of E-cadherin.
abstract: Self-renewal and differentiation of embryonic stem cells are tightly coordinated with cell-cycle progression and reconstructions. However, technical approach to directly visualize single embryonic stem cells still remains challenging. Here we combined two independent systems by using artificially constructed extracellular matrix that maintains embryonic stem cells in single level with cell cycle visualization reporters to directly observe cell cycle progression. Using Fucci (fluorescent ubiquitination-based cell cycle indicator) technology and computer-assisted fluorescence microscopy we were able to visualize cell cycle progression of mouse embryonic stem cells prepared from Fucci2 knock-in mice (mES/Fucci2). Imaged mES/Fucci2 cells were plated on coverslips coated with recombinant E-cadherin-IgG Fc (E-cad-Fc). This artificial extracellular matrix effectively increases adherence of cultured cells to coverslips, which is advantageous for fluorescence imaging. mES/Fucci2 cells on the E-cad-Fc maintained the typical cell cycle of mES cells with truncated G1 phase and pluripotency. During time-lapse imaging, we were able to track these cells with dendritic-like cell morphology and many pseudopodial protrusions. By contrast, the cell cycle progression of mES/Fucci2 cells on mouse embryonic fibroblasts (MEFs) was not observable due to their compact aggregation. Cell cycle duration of mES/Fucci2 cells on the E-cad-Fc was 16 h. Thus, the unique properties of our immunocytochemical analysis have revealed that decline of pluripotency of the Fucci2 mES cells on the E-cad-Fc was coordinated with their differentiation.
authors: Department of Biomolecular Engineering, Tokyo Institute of Technology, Yokohama, 226-8501 Japan.
authors: Dragomirka
authors: D
authors: Jovic
authors: Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198 Japan.
authors: Asako
authors: A
authors: Sakaue-Sawano
authors: Laboratory for Animal Resources and Genetic Engineering, Riken CDB, Kobe, 650-0047 Japan.
authors: Takaya
authors: T
authors: Abe
authors: Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, 151-921 Republic of Korea.
authors: Chong-Su
authors: CS
authors: Cho
authors: Department of Biomolecular Engineering, Tokyo Institute of Technology, Yokohama, 226-8501 Japan ; Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226 USA.
authors: Masato
authors: M
authors: Nagaoka
authors: Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198 Japan.
authors: Atsushi
authors: A
authors: Miyawaki
authors: Frontier Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama, 226 8501 Japan.
authors: Toshihiro
authors: T
authors: Akaike
keywords: Cell cycle; E-cad-Fc; Embryonic stem cells; Fucci; Live imaging