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Direct observation of cell cycle progression in living mouse embryonic stem cells on an extracellular matrix of E-cadherin

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 co...

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Autores principales: Jovic, Dragomirka, Sakaue-Sawano, Asako, Abe, Takaya, Cho, Chong-Su, Nagaoka, Masato, Miyawaki, Atsushi, Akaike, Toshihiro
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer International Publishing 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320234/
https://www.ncbi.nlm.nih.gov/pubmed/25674414
http://dx.doi.org/10.1186/2193-1801-2-585
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author Jovic, Dragomirka
Sakaue-Sawano, Asako
Abe, Takaya
Cho, Chong-Su
Nagaoka, Masato
Miyawaki, Atsushi
Akaike, Toshihiro
author_facet Jovic, Dragomirka
Sakaue-Sawano, Asako
Abe, Takaya
Cho, Chong-Su
Nagaoka, Masato
Miyawaki, Atsushi
Akaike, Toshihiro
author_sort Jovic, Dragomirka
collection PubMed
description 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 G(1) 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/2193-1801-2-585) contains supplementary material, which is available to authorized users.
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spelling pubmed-43202342015-02-11 Direct observation of cell cycle progression in living mouse embryonic stem cells on an extracellular matrix of E-cadherin Jovic, Dragomirka Sakaue-Sawano, Asako Abe, Takaya Cho, Chong-Su Nagaoka, Masato Miyawaki, Atsushi Akaike, Toshihiro Springerplus Research 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 G(1) 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/2193-1801-2-585) contains supplementary material, which is available to authorized users. Springer International Publishing 2013-10-31 /pmc/articles/PMC4320234/ /pubmed/25674414 http://dx.doi.org/10.1186/2193-1801-2-585 Text en © Jovic et al.; licensee Springer. 2013 This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research
Jovic, Dragomirka
Sakaue-Sawano, Asako
Abe, Takaya
Cho, Chong-Su
Nagaoka, Masato
Miyawaki, Atsushi
Akaike, Toshihiro
Direct observation of cell cycle progression in living mouse embryonic stem cells on an extracellular matrix of E-cadherin
title Direct observation of cell cycle progression in living mouse embryonic stem cells on an extracellular matrix of E-cadherin
title_full Direct observation of cell cycle progression in living mouse embryonic stem cells on an extracellular matrix of E-cadherin
title_fullStr Direct observation of cell cycle progression in living mouse embryonic stem cells on an extracellular matrix of E-cadherin
title_full_unstemmed Direct observation of cell cycle progression in living mouse embryonic stem cells on an extracellular matrix of E-cadherin
title_short Direct observation of cell cycle progression in living mouse embryonic stem cells on an extracellular matrix of E-cadherin
title_sort direct observation of cell cycle progression in living mouse embryonic stem cells on an extracellular matrix of e-cadherin
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320234/
https://www.ncbi.nlm.nih.gov/pubmed/25674414
http://dx.doi.org/10.1186/2193-1801-2-585
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