Myocardin Overexpression Is Sufficient for Promoting the Development of a Mature Smooth Muscle Cell-Like Phenotype from Human Embryonic Stem Cells
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{"title"=>"Myocardin Overexpression Is Sufficient for Promoting the Development of a Mature Smooth Muscle Cell-Like Phenotype from Human Embryonic Stem Cells", "type"=>"journal", "authors"=>[{"first_name"=>"Linda", "last_name"=>"Raphel", "scopus_author_id"=>"55346686700"}, {"first_name"=>"Amarnath", "last_name"=>"Talasila", "scopus_author_id"=>"26032444400"}, {"first_name"=>"Christine", "last_name"=>"Cheung", "scopus_author_id"=>"53867867900"}, {"first_name"=>"Sanjay", "last_name"=>"Sinha", "scopus_author_id"=>"34975904500"}], "year"=>2012, "source"=>"PLoS ONE", "identifiers"=>{"scopus"=>"2-s2.0-84865520545", "sgr"=>"84865520545", "issn"=>"19326203", "doi"=>"10.1371/journal.pone.0044052", "pmid"=>"22937150", "isbn"=>"1932-6203 (Electronic)\\r1932-6203 (Linking)", "pui"=>"365529065"}, "id"=>"e2bf8aa5-caf9-3388-93bd-1a732c6478f7", "abstract"=>"BACKGROUND: Myocardin is thought to have a key role in smooth muscle cell (SMC) development by acting on CArG-dependent genes. However, it is unclear whether myocardin-induced SMC maturation and increases in agonist-induced calcium signalling are also associated with increases in the expression of non-CArG-dependent SMC-specific genes. Moreover, it is unknown whether myocardin promotes SMC development from human embryonic stem cells.\\n\\nMETHODOLOGY/PRINCIPAL: Findings The effects of adenoviral-mediated myocardin overexpression on SMC development in human ESC-derived embryoid bodies were investigated using immunofluorescence, flow cytometry and real time RT-PCR. Myocardin overexpression from day 10 to day 28 of embryoid body differentiation increased the number of smooth muscle α-actin(+) and smooth muscle myosin heavy chain(+) SMC-like cells and increased carbachol-induced contractile function. However, myocardin was found to selectively regulate only CArG-dependent SMC-specific genes. Nevertheless, myocardin expression appeared to be sufficient to specify the SMC lineage.\\n\\nCONCLUSIONS/SIGNIFICANCE: Myocardin increases the development and maturation of SMC-like cells from human embryonic stem cells despite not activating the full repertoire of SMC genes. These findings have implications for vascular tissue engineering and other applications requiring large numbers of functional SMCs.", "link"=>"http://www.mendeley.com/research/myocardin-overexpression-sufficient-promoting-development-mature-smooth-muscle-celllike-phenotype-hu", "reader_count"=>19, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Researcher"=>5, "Student > Ph. D. Student"=>8, "Student > Postgraduate"=>2, "Lecturer > Senior Lecturer"=>1, "Professor"=>2}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Researcher"=>5, "Student > Ph. D. Student"=>8, "Student > Postgraduate"=>2, "Lecturer > Senior Lecturer"=>1, "Professor"=>2}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>3, "Agricultural and Biological Sciences"=>11, "Medicine and Dentistry"=>4}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>4}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>11}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>3}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"United States"=>1}, "group_count"=>0}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/585670"], "description"=>"<p>Primer sequences used for real time RT-PCR.</p>", "links"=>[], "tags"=>["sequences"], "article_id"=>256161, "categories"=>["Physiology", "Molecular Biology", "Cell Biology", "Developmental Biology"], "users"=>["Linda Raphel", "Amarnath Talasila", "Christine Cheung", "Sanjay Sinha"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044052.t001", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Primer_sequences_used_for_real_time_RT_PCR_/256161", "title"=>"Primer sequences used for real time RT-PCR.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2012-08-28 01:42:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/585520"], "description"=>"<p>Embryoid bodies were treated with Ad-LacZ or Ad-Myo from day 10 to day 28 then dispersed and fixed for flow cytometry. (A) The subset of cells transduced with the Ad-Myo virus was identified by flow cytometric detection of the 3′ FLAG tag fused to the myocardin transgene. (B) The effect of Ad-Myo treatment on %SMMHC<sup>+</sup> cells was analysed by flow cytometry. The majority (90%) of FLAG<sup>+</sup> Ad-Myo transduced cells demonstrated a SMC-like phenotype. Data are representative of three independent experiments.</p>", "links"=>[], "tags"=>["instructive", "smc-like", "cells", "embryoid"], "article_id"=>256004, "categories"=>["Physiology", "Molecular Biology", "Cell Biology", "Developmental Biology"], "users"=>["Linda Raphel", "Amarnath Talasila", "Christine Cheung", "Sanjay Sinha"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044052.g007", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Myocardin_has_a_dominant_instructive_role_on_development_of_SMC_like_cells_in_the_embryoid_body_/256004", "title"=>"Myocardin has a dominant instructive role on development of SMC-like cells in the embryoid body.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-28 01:40:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/307637", "https://ndownloader.figshare.com/files/307899", "https://ndownloader.figshare.com/files/308155", "https://ndownloader.figshare.com/files/308209", "https://ndownloader.figshare.com/files/308417"], "description"=>"<div><h3>Background</h3><p>Myocardin is thought to have a key role in smooth muscle cell (SMC) development by acting on CArG-dependent genes. However, it is unclear whether myocardin-induced SMC maturation and increases in agonist-induced calcium signalling are also associated with increases in the expression of non-CArG-dependent SMC-specific genes. Moreover, it is unknown whether myocardin promotes SMC development from human embryonic stem cells.</p> <h3>Methodology/Principal</h3><p>Findings The effects of adenoviral-mediated myocardin overexpression on SMC development in human ESC-derived embryoid bodies were investigated using immunofluorescence, flow cytometry and real time RT-PCR. Myocardin overexpression from day 10 to day 28 of embryoid body differentiation increased the number of smooth muscle α-actin<sup>+</sup> and smooth muscle myosin heavy chain<sup>+</sup> SMC-like cells and increased carbachol-induced contractile function. However, myocardin was found to selectively regulate only CArG-dependent SMC-specific genes. Nevertheless, myocardin expression appeared to be sufficient to specify the SMC lineage.</p> <h3>Conclusions/Significance</h3><p>Myocardin increases the development and maturation of SMC-like cells from human embryonic stem cells despite not activating the full repertoire of SMC genes. These findings have implications for vascular tissue engineering and other applications requiring large numbers of functional SMCs.</p> </div>", "links"=>[], "tags"=>["myocardin", "overexpression", "promoting", "mature", "cell-like", "phenotype", "embryonic", "cells"], "article_id"=>120709, "categories"=>["Physiology", "Molecular Biology", "Cell Biology", "Developmental Biology"], "users"=>["Linda Raphel", "Amarnath Talasila", "Christine Cheung", "Sanjay Sinha"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0044052.s001", "https://dx.doi.org/10.1371/journal.pone.0044052.s002", "https://dx.doi.org/10.1371/journal.pone.0044052.s003", "https://dx.doi.org/10.1371/journal.pone.0044052.s004", "https://dx.doi.org/10.1371/journal.pone.0044052.s005"], "stats"=>{"downloads"=>1, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Myocardin_Overexpression_Is_Sufficient_for_Promoting_the_Development_of_a_Mature_Smooth_Muscle_Cell_Like_Phenotype_from_Human_Embryonic_Stem_Cells/120709", "title"=>"Myocardin Overexpression Is Sufficient for Promoting the Development of a Mature Smooth Muscle Cell-Like Phenotype from Human Embryonic Stem Cells", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2012-08-28 00:11:49"}
  • {"files"=>["https://ndownloader.figshare.com/files/585228"], "description"=>"<p>Embryoid bodies were treated with no virus, Ad-LacZ or Ad-Myo from day 10 to day 28 and then harvested for RNA. SMC marker expression for a range of CArG-dependent and non-dependent genes was measured using real time RT-PCR and is normalised by three housekeeping genes and then presented relative to no virus controls. RT-PCR data represent means from at least three independent experiments. Error bars represent s.e.m. SMαA and SMMHC expression levels in response to myocardin were significantly higher than the other genes and thus the precise levels are depicted by numbers above the black bars (± s.e.m.).</p>", "links"=>[], "tags"=>["upregulation", "carg-dependent", "genes"], "article_id"=>255720, "categories"=>["Physiology", "Molecular Biology", "Cell Biology", "Developmental Biology"], "users"=>["Linda Raphel", "Amarnath Talasila", "Christine Cheung", "Sanjay Sinha"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044052.g004", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Selective_upregulation_of_CArG_dependent_genes_by_myocardin_/255720", "title"=>"Selective upregulation of CArG-dependent genes by myocardin.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-28 01:35:20"}
  • {"files"=>["https://ndownloader.figshare.com/files/585611"], "description"=>"<p>Embryoid bodies were transduced with Ad-LacZ or Ad-Myo early (day 10 alone or days 10 & 14), late (days 18 & 23) or throughout differentiation (days 10, 14, 18 &13). Late delivery of Ad-Myo reduced number of SMαA<sup>+</sup> cells (A) (*p<0.05 by ANOVA and Tukey HSD) but had no significant effect on SMMHC<sup>+</sup> cell numbers (B). Values represent mean cell proportions from three independent experiments (± s.e.m.). *p<0.05.</p>", "links"=>[], "tags"=>["viral", "transduction", "reveals", "smc", "induction"], "article_id"=>256094, "categories"=>["Physiology", "Molecular Biology", "Cell Biology", "Developmental Biology"], "users"=>["Linda Raphel", "Amarnath Talasila", "Christine Cheung", "Sanjay Sinha"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044052.g008", "stats"=>{"downloads"=>1, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Timing_of_viral_transduction_reveals_an_early_role_in_SMC_induction_for_myocardin_/256094", "title"=>"Timing of viral transduction reveals an early role in SMC induction for myocardin.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-28 01:41:34"}
  • {"files"=>["https://ndownloader.figshare.com/files/584784"], "description"=>"<p>(A) Low power dark field image of day 28 human embryoid body grown in 20% FBS showing outgrowth of cells (white arrowheads) from the central embryoid body mass. (B and C) Expression of SMC specific genes in embryoid bodies by real time RT-PCR at days 0, 15 and 28 is normalised by three housekeeping genes (GAPDH, UBC, 18S) and then presented relative to undifferentiated human ESCs. RT-PCR data represent means from three independent experiments. Bars represent s.e.m. Cells that stain for SMαA (D) and SMMHC (E) clearly seen at day 28 within embryoid bodies by immunofluorescence. Nuclei counterstained (blue) with DAPI. Bar in A = 1000 µm, bars in D and E = 20 µm.</p>", "links"=>[], "tags"=>["smcs", "embryoid"], "article_id"=>255278, "categories"=>["Physiology", "Molecular Biology", "Cell Biology", "Developmental Biology"], "users"=>["Linda Raphel", "Amarnath Talasila", "Christine Cheung", "Sanjay Sinha"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044052.g001", "stats"=>{"downloads"=>0, "page_views"=>2, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Development_of_SMCs_in_human_embryoid_bodies_/255278", "title"=>"Development of SMCs in human embryoid bodies.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-28 01:27:58"}
  • {"files"=>["https://ndownloader.figshare.com/files/585432"], "description"=>"<p>Embryoid bodies were treated with Ad-LacZ or Ad-Myo from day 10 to day 28, dispersed into a single cell suspension and seeded into collagen gels in 24 well plates (A). Gel contraction in response to carbachol was significantly increased in the Ad-myo group (B). Data points represent the means (±s.e.m.) of three experiments. SMCs derived from human ESCs using a 2-dimensional culture protocol were individually examined for contraction following transduction with Ad-LacZ or Ad-Myo using time lapse microscopy. Percentage of contractile cells increased from 29% with Ad-LacZ to 53% with Ad-Myo (C). Results represent the mean values (±s.e.m.) from 10 randomly chosen optical fields. **p<0.01.</p>", "links"=>[], "tags"=>["phenotype", "promoted", "myocardin"], "article_id"=>255921, "categories"=>["Physiology", "Molecular Biology", "Cell Biology", "Developmental Biology"], "users"=>["Linda Raphel", "Amarnath Talasila", "Christine Cheung", "Sanjay Sinha"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044052.g006", "stats"=>{"downloads"=>0, "page_views"=>4, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Contractile_phenotype_is_promoted_by_myocardin_overexpression_/255921", "title"=>"Contractile phenotype is promoted by myocardin overexpression.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-28 01:38:41"}
  • {"files"=>["https://ndownloader.figshare.com/files/585064"], "description"=>"<p>Embryoid bodies were enzymatically dispersed into single cells at day 17 or day 28 and flow cytometric assessment for SMC markers was carried out. Groups that had been treated with no virus, Ad-LacZ or Ad-Myo from day 10 onwards were used to quantify the proportion of SMαA<sup>+</sup> cells (A & B) and SMMHC<sup>+</sup> cells (C & D). Both FL1 and FL2 channels were measured for all samples to distinguish specific signal for SMαA (FL1 in A) and SMMHC (FL2 in B) due to the high levels of autofluorescence in embryoid body-derived cells. In the no virus group, SMαA staining was quantified as median SMαA<sup>+</sup> signal/median SMαA<sup>−</sup> signal at both day 17 and day 28 (E). Data presented in A and C are representative flow cytometric plots from a single study with the means from three independent experiments specified in the gated regions and as bar charts ± s.e.m. (B, D & E). **p<0.01.</p>", "links"=>[], "tags"=>["overexpression", "increases", "smc-like"], "article_id"=>255550, "categories"=>["Physiology", "Molecular Biology", "Cell Biology", "Developmental Biology"], "users"=>["Linda Raphel", "Amarnath Talasila", "Christine Cheung", "Sanjay Sinha"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044052.g003", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Myocardin_overexpression_increases_number_of_SMC_like_cells_/255550", "title"=>"Myocardin overexpression increases number of SMC-like cells.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-28 01:32:30"}
  • {"files"=>["https://ndownloader.figshare.com/files/584932"], "description"=>"<p>Immunoblotting of embryoid bodies transduced with Ad-LacZ or Ad-Myo confirm myocardin overexpression at a variety of developmental times (A). Immunofluorescent assessment of day 28 embryoid bodies reveals an increase in SMαA, calponin and SMMHC (B) staining following treatment with Ad-Myo from day 10 onwards, in comparison to no treatment or Ad-LacZ negative controls. Photomicrograph exposure times were kept constant across all three treatment groups and deliberately low enough to clearly show the large increases in fluorescent intensity in the Ad-Myo group. Nuclei counterstained (blue) with DAPI. Bar = 100 µm.</p>", "links"=>[], "tags"=>["overexpression", "increases", "calponin", "smmhc"], "article_id"=>255414, "categories"=>["Physiology", "Molecular Biology", "Cell Biology", "Developmental Biology"], "users"=>["Linda Raphel", "Amarnath Talasila", "Christine Cheung", "Sanjay Sinha"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044052.g002", "stats"=>{"downloads"=>1, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Myocardin_overexpression_increases_SM_945_A_calponin_and_SMMHC_staining_/255414", "title"=>"Myocardin overexpression increases SMαA, calponin and SMMHC staining.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-28 01:30:14"}
  • {"files"=>["https://ndownloader.figshare.com/files/585316"], "description"=>"<p>Embryoid bodies were treated with Ad-LacZ or Ad-Myo from day 10 to day 28 and then dispersed into a single cell suspension. Cells were loaded with Fluo-4, a calcium sensitive fluorophore, and intracellular [Ca<sup>2+</sup>] was measured by flow cytometry before and after the addition of the muscarinic agonist, carbachol in arbitrary units (AU). Representative data from a single experiment (A) and means of three studies (B) show significant increases in fluorescence following addition of carbachol. Error bars represent s.e.m., ns = not significant, * = p<0.05, C = carbachol.</p>", "links"=>[], "tags"=>["calcium", "influx"], "article_id"=>255806, "categories"=>["Physiology", "Molecular Biology", "Cell Biology", "Developmental Biology"], "users"=>["Linda Raphel", "Amarnath Talasila", "Christine Cheung", "Sanjay Sinha"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0044052.g005", "stats"=>{"downloads"=>3, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Myocardin_promotes_calcium_influx_in_response_to_carbachol_/255806", "title"=>"Myocardin promotes calcium influx in response to carbachol.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2012-08-28 01:36:46"}

PMC Usage Stats | Further Information

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Relative Metric

{"start_date"=>"2012-01-01T00:00:00Z", "end_date"=>"2012-12-31T00:00:00Z", "subject_areas"=>[{"subject_area"=>"/Biology and life sciences", "average_usage"=>[322, 550, 671, 773, 864, 955, 1048, 1135, 1223, 1308, 1387, 1465, 1534, 1602, 1673, 1744, 1813, 1885, 1955, 2026, 2093, 2160, 2228, 2290, 2349]}, {"subject_area"=>"/Biology and life sciences/Developmental biology", "average_usage"=>[313, 562, 690, 796, 893, 986, 1079, 1173, 1257, 1349, 1429, 1506, 1586, 1661, 1730, 1803, 1876, 1945, 2017, 2091, 2163, 2225, 2294, 2362, 2430]}]}
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