Rho Kinases Regulate the Renewal and Neural Differentiation of Embryonic Stem Cells in a Cell Plating Density–Dependent Manner
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{"title"=>"Rho kinases regulate the renewal and neural differentiation of embryonic stem cells in a cell plating density-dependent manner", "type"=>"journal", "authors"=>[{"first_name"=>"Tzu Ching", "last_name"=>"Chang", "scopus_author_id"=>"23977577700"}, {"first_name"=>"Yen Chung", "last_name"=>"Chen", "scopus_author_id"=>"8895108300"}, {"first_name"=>"Ming Hua", "last_name"=>"Yang", "scopus_author_id"=>"55703261200"}, {"first_name"=>"Chien Hung", "last_name"=>"Chen", "scopus_author_id"=>"57129751200"}, {"first_name"=>"En Wei", "last_name"=>"Hsing", "scopus_author_id"=>"36092889000"}, {"first_name"=>"Bor Sheng", "last_name"=>"Ko", "scopus_author_id"=>"35210620900"}, {"first_name"=>"Jun Yang", "last_name"=>"Liou", "scopus_author_id"=>"7202248728"}, {"first_name"=>"Kenneth K.", "last_name"=>"Wu", "scopus_author_id"=>"7404512056"}], "year"=>2010, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"20169147", "doi"=>"10.1371/journal.pone.0009187", "sgr"=>"77949437184", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "scopus"=>"2-s2.0-77949437184", "issn"=>"19326203", "pui"=>"358460783"}, "id"=>"45952b0b-f2af-3711-b815-64796d436073", "abstract"=>"BACKGROUND: Rho kinases (ROCKs) mediate cell contraction, local adhesion, and cell motility, which are considered to be important in cell differentiation. We postulated that ROCKs are involved in controlling embryonic stem (ES) cell renewal and differentiation.\\n\\nMETHODOLOGY/PRINCIPAL FINDINGS: CCE, a murine ES cell, was treated with Y-27632 for 48 to 96 hours and colony formation was evaluated. Y-27632 blocked CCE colony formation and induced CCE to grow as individual cells, regardless of the initial seeding cell density either at 10(4)/cm(2) (\"high\" seeding density) or 2x10(3)/cm(2) (\"low\" density). However, at high seeding density, Y-27632-treated cells exhibited reduction of alkaline phosphatase (AP) staining and Oct3/4 expression. They expressed SOX-1, nestin, and MAP2c, but not betaIII-tubulin or NG-2. They did not express endoderm or mesoderm lineage markers. After removal of Y-27632, the cells failed to form colonies or regain undifferentiated state. Silencing of ROCK-1 or ROCK-2 with selective small interference RNA induced CCE morphological changes similar to Y-27632. Silencing of ROCK-1 or ROCK-2 individually was sufficient to cause reduction of AP and Oct3/4, and expression of SOX-1, nestin, and MAP2c; and combined silencing of both ROCKs did not augment the effects exerted by individual ROCK siRNA. Y-27632-treated CCE cells seeded at 2x10(3) or 6.6x10(3) cells/cm(2) did not lose renewal factors or express differentiation markers. Furthermore, they were able to form AP-positive colonies after removal of Y-27632 and reseeding. Similar to ROCK inhibition by Y-27632, silencing of ROCK-1 or ROCK-2 in cells seeded at 2x10(3)/cm(2) did not change renewal factors.\\n\\nCONCLUSIONS/SIGNIFICANCE: We conclude that ROCKs promote ES cell colony formation, maintain them at undifferentiated state, and prevent them from neural differentiation at high seeding density. ROCK inhibition represents a new strategy for preparing large numbers of neural progenitor cells.", "link"=>"http://www.mendeley.com/research/rho-kinases-regulate-renewal-neural-differentiation-embryonic-stem-cells-cell-plating-densitydepende", "reader_count"=>46, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>3, "Researcher"=>14, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>14, "Student > Master"=>5, "Other"=>1, "Student > Bachelor"=>2, "Lecturer"=>1, "Professor"=>3}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>3, "Researcher"=>14, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>14, "Student > Master"=>5, "Other"=>1, "Student > Bachelor"=>2, "Lecturer"=>1, "Professor"=>3}, "reader_count_by_subject_area"=>{"Engineering"=>4, "Unspecified"=>3, "Biochemistry, Genetics and Molecular Biology"=>1, "Materials Science"=>2, "Agricultural and Biological Sciences"=>25, "Medicine and Dentistry"=>8, "Neuroscience"=>2, "Earth and Planetary Sciences"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>4}, "Materials Science"=>{"Materials Science"=>2}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>8}, "Neuroscience"=>{"Neuroscience"=>2}, "Earth and Planetary Sciences"=>{"Earth and Planetary Sciences"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>25}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}, "Unspecified"=>{"Unspecified"=>3}}, "reader_count_by_country"=>{"United States"=>2, "Japan"=>1, "United Kingdom"=>1, "Australia"=>1, "France"=>1, "Spain"=>1}, "group_count"=>0}

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  • {"files"=>["https://ndownloader.figshare.com/files/862917"], "description"=>"<p><b><i>A.</i></b> CCE cells seeded at 10<sup>4</sup> cells/cm<sup>2</sup> for 24 h were treated with Y-27632 (10 µM) or RA (10 µM) for 48 h. ROCK-1 and ROCK-2 proteins were analyzed by Western blotting. <b><i>B.</i></b> ROCK activities in CCE cells treated with Y-27632 or H-89 (10 µM) for 2 h. Each bar denotes mean ± SEM of three experiments.</p>", "links"=>[], "tags"=>["proteins", "activities", "cce", "cells", "y-27632"], "article_id"=>533358, "categories"=>["Computational Biology", "Cell Biology", "Medicine"], "users"=>["Tzu-Ching Chang", "Yen-Chung Chen", "Ming-Hua Yang", "Chien-Hung Chen", "En-Wei Hsing", "Bor-Sheng Ko", "Jun-Yang Liou", "Kenneth K. Wu"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0009187.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_ROCK_proteins_and_activities_in_CCE_cells_with_amp_without_Y_27632_treatment_/533358", "title"=>"ROCK proteins and activities in CCE cells with & without Y-27632 treatment.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 02:39:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/863034"], "description"=>"<p><b><i>A & B.</i></b> Cells in suspension were treated with Y-27632 or RA and seeded at 2×10<sup>3</sup> cells/cm<sup>2</sup> for 96 h (<b>A</b>) or 1×10<sup>4</sup> cells/cm<sup>2</sup> for 48 h (<b><i>B</i></b>). <b><i>C.</i></b> Cell suspensions were seeded at 2×10<sup>3</sup> cells/cm<sup>2</sup> for 24 h followed by treatment with the indicated agent for 96 h. <b><i>D.</i></b> Cell suspensions seeded at 1×10<sup>4</sup> cells/cm<sup>2</sup> for 24 h were treated with the indicated agent for 48 h. Cells were examined under phase microscopy.</p>", "links"=>[], "tags"=>["y-27632", "cce", "morphological"], "article_id"=>533486, "categories"=>["Computational Biology", "Cell Biology", "Medicine"], "users"=>["Tzu-Ching Chang", "Yen-Chung Chen", "Ming-Hua Yang", "Chien-Hung Chen", "En-Wei Hsing", "Bor-Sheng Ko", "Jun-Yang Liou", "Kenneth K. Wu"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0009187.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_Y_27632_on_CCE_colony_formation_and_morphological_changes_/533486", "title"=>"Effect of Y-27632 on CCE colony formation and morphological changes.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 02:39:43"}
  • {"files"=>["https://ndownloader.figshare.com/files/863193"], "description"=>"<p>CCE cells seeded at 1×10<sup>4</sup> cells/cm<sup>2</sup> for 24 h were treated with Y-27632 or other agents for 48 h and cells were analyzed with AP staining (<b><i>A</i></b>), Oct3/4 immunofluorescence (<b><i>B</i></b>) and Oct3/4 Western blotting (<b><i>C</i></b>). The blots in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009187#pone-0009187-g003\" target=\"_blank\">Figure 3C</a> were quantitated by densitometry. Each bar denotes mean ± SEM of three experiments. PI denotes propidium iodide staining and phase, phase contrast microscopy.</p>", "links"=>[], "tags"=>["ap"], "article_id"=>533645, "categories"=>["Computational Biology", "Cell Biology", "Medicine"], "users"=>["Tzu-Ching Chang", "Yen-Chung Chen", "Ming-Hua Yang", "Chien-Hung Chen", "En-Wei Hsing", "Bor-Sheng Ko", "Jun-Yang Liou", "Kenneth K. Wu"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0009187.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Reduction_of_AP_and_Oct3_4_by_ROCK_inhibition_/533645", "title"=>"Reduction of AP and Oct3/4 by ROCK inhibition.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 02:40:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/863318"], "description"=>"<p><b><i>A.</i></b> Western blot analysis of renewal factors in CCE cells seeded at low-density (2×10<sup>3</sup> cells/cm<sup>2</sup>) for 24 h followed by treatment with Y-27632 (10 µM), RA (10 µM) or DMSO vehicle for 48 h and 96 h. <b><i>B.</i></b> Western blot analysis of renewal factors in CCE cells seeded at 6.6×10<sup>3</sup> vs. 2×10<sup>3</sup> cells/cm<sup>2</sup> for 24 h and treated with inhibitors for 96 h. <b><i>C</i></b><b>.</b> Low-density CCE cells were treated with vehicle control or Y-27632 for 96 h, harvested and reseeded at 2×10<sup>3</sup> cells/cm<sup>2</sup> for 48 h. Cells were stained for AP.</p>", "links"=>[], "tags"=>["renewal", "properties", "seeding"], "article_id"=>533770, "categories"=>["Computational Biology", "Cell Biology", "Medicine"], "users"=>["Tzu-Ching Chang", "Yen-Chung Chen", "Ming-Hua Yang", "Chien-Hung Chen", "En-Wei Hsing", "Bor-Sheng Ko", "Jun-Yang Liou", "Kenneth K. Wu"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0009187.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Retaining_of_renewal_properties_at_low_seeding_density_/533770", "title"=>"Retaining of renewal properties at low seeding density.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 02:41:15"}
  • {"files"=>["https://ndownloader.figshare.com/files/863438"], "description"=>"<p>CCE cells seeded at 1×10<sup>4</sup> cells/cm<sup>2</sup> for 24 h were treated with the indicated inhibitors for 48 h. <b><i>A</i></b>. Nestin and other NPC proteins were analyzed by Western blotting. <b><i>B</i></b>. Nestin mRNA levels were analyzed by quantitative PCR. Each bar denotes mean ± SEM of three experiments. <b><i>C</i></b>. Neuronal markers and <b><i>D</i></b>. GATA-4 and FIK-1 proteins were analyzed by Western blotting. The blots are representative of those from three experiments.</p>", "links"=>[], "tags"=>["npc", "markers", "high-density", "cce", "cells", "treated"], "article_id"=>533883, "categories"=>["Computational Biology", "Cell Biology", "Medicine"], "users"=>["Tzu-Ching Chang", "Yen-Chung Chen", "Ming-Hua Yang", "Chien-Hung Chen", "En-Wei Hsing", "Bor-Sheng Ko", "Jun-Yang Liou", "Kenneth K. Wu"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0009187.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Expression_of_NPC_markers_in_high_density_CCE_cells_treated_with_ROCK_inhibitors_/533883", "title"=>"Expression of NPC markers in high-density CCE cells treated with ROCK inhibitors.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 02:41:56"}
  • {"files"=>["https://ndownloader.figshare.com/files/863659"], "description"=>"<p><b><i>A</i></b>. CCE cells transfected with selective ROCK-1 and ROCK-2 siRNA or control RNA were seeded at 10<sup>4</sup> cells/cm<sup>2</sup> for 48 h, and ROCK-1 and ROCK-2 proteins were analyzed by Western blotting. The blots were quantitated by densitometry. Each bar denotes mean ± SEM (n = 3). <b><i>B.</i></b> Colony formation and growing pattern were examined under phase-contrast microscopy. <b><i>C</i></b>. Cells at 1×10<sup>4</sup> cells/cm<sup>2</sup> were treated with ROCK-1 plus ROCK-2 siRNA vs. individual ROCK siRNA. Oct3/4 proteins were analyzed by Western blotting. <b><i>D.</i></b> Oct3/4 and SOX-2 proteins were analyzed in siRNA-treated cells seeded at 1×10<sup>4</sup> cells/cm<sup>2</sup> (left panel) vs. 2×10<sup>3</sup> cells/cm<sup>2</sup> (right panel). The blots were analyzed by densitometry. Each bar denotes mean ± SEM (n = 3).</p>", "links"=>[], "tags"=>["sirna", "renewal"], "article_id"=>534106, "categories"=>["Computational Biology", "Cell Biology", "Medicine"], "users"=>["Tzu-Ching Chang", "Yen-Chung Chen", "Ming-Hua Yang", "Chien-Hung Chen", "En-Wei Hsing", "Bor-Sheng Ko", "Jun-Yang Liou", "Kenneth K. Wu"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0009187.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Influence_of_ROCK_siRNA_on_colony_formation_and_renewal_factors_/534106", "title"=>"Influence of ROCK siRNA on colony formation and renewal factors.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 02:43:08"}
  • {"files"=>["https://ndownloader.figshare.com/files/863833"], "description"=>"<p><b><i>A</i></b>. CCE cells were seeded at 1×10<sup>4</sup> cells/cm<sup>2</sup> for 48 h and SOX-1, nestin and MAP2c proteins in CCE cell lysates were analyzed by Western blotting. <b><i>B</i></b>. CCE cells were seeded at 2×10<sup>3</sup> cells/cm<sup>2</sup> for 48 h and nestin proteins were analyzed by Western blotting. The blot was quantitated with densitometry. Each bar denotes mean ± SEM (n = 3).</p>", "links"=>[], "tags"=>["npc", "markers", "sirna", "density-dependent"], "article_id"=>534280, "categories"=>["Computational Biology", "Cell Biology", "Medicine"], "users"=>["Tzu-Ching Chang", "Yen-Chung Chen", "Ming-Hua Yang", "Chien-Hung Chen", "En-Wei Hsing", "Bor-Sheng Ko", "Jun-Yang Liou", "Kenneth K. Wu"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0009187.g007"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Regulation_of_NPC_markers_by_ROCK_siRNA_in_a_cell_density_dependent_manner_/534280", "title"=>"Regulation of NPC markers by ROCK siRNA in a cell density-dependent manner.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2013-02-21 02:44:10"}

PMC Usage Stats | Further Information

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