Journal: BMC Genomics

Article Title: Gene expression analysis of embryonic stem cells expressing VE-cadherin (CD144) during endothelial differentiation

doi: 10.1186/1471-2164-9-240

Figure Lengend Snippet: CD41 and CD144 during ES cell differentiation . ( A ) Flow cytometric analysis of stem cell differentiation. CCE murine ES cells differentiated into cystic embryoid bodies after removal of LIF (see methods). Flow cytometry was used to determine the expression of VEGF-R2, CD144, and CD41. VEGF-R2 positve cells were first detected at 2.5 days after the removal of LIF and represent approximately 11% of the cells. CD144 and CD41 are first detected at day 3.5. Both markers, CD144 and CD41, represent a subset of VEGF-R2 positive cells (4.4 and 5.5%, respectively). Only a small percentage of CD144 positive cells (1.7%) co-express CD41 at day 3.5 (bottom right panel). Isotype controls are shown in the upper right panel. ( B ) CD144 and CD41-expressing cells remain a subset of VEGF-R2 positive cells (5.2 and 3.8%, respectively) at day 6.5, however, at this time point, the two cell markers are expressed on different cell populations (lower right panel). ( C ) A schematic of ES cell differentiation demonstrating the populations of cells isolated for microarray analysis. VEGF-R2 is a marker of the hemangioblast (HB). CD41 is one of the earliest markers for the hematopoietic progenitor (HP) cells and can be detected as early as day 3.5, concurrently with the endothelial-specific marker CD144 (VE-cadherin) expressed on the angioblast (AB) at day 3.5, and also on differentiated endothelial cells (ET) at day 6.5. ( D ) Protein expression of CD144 (VE-cadherin) and CD41 in embryonic stem cell differentiation. Isolated proteins from developing stem cells on days 2.5 to 8.5 were separated on a 10% SDS-PAGE. Western blot analysis was performed with antibodies directed against CD144, CD41, and β-actin.

Article Snippet: CCE Embryonic stem cells (ATCC) were maintained on irradiated primary embryonic fibroblasts (Chemicon) in knockout DMEM (Invitrogen/Gibco-BRL) supplemented with 15% fetal bovine serum (Hyclone, Logan, UT), penicillin/streptomycin 1% (Invitrogen/Gibco-BRL), l-glutamine 2 mM (Invitrogen/Gibco-BRL), non-essential amino acids 0.1 mM, nucleosides 0.1 mM, 2-mercaptoethanol 0.1 mM (Sigma, St. Louis, MO), monothioglycerol (MTG) 2 mM (Sigma), and ESGRO leukemia inhibitory factor (LIF) 1000 units/ml (Sigma).

Techniques: Cell Differentiation, Flow Cytometry, Expressing, Isolation, Microarray, Marker, SDS Page, Western Blot

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: Traction forces are necessary for definitive endoderm specification. Mouse ESCs (CCE and R1) were grown on decellularized fibroblast-derived ECM in definitive endoderm induction medium, with or without 10 μM blebbistatin. (A) Immunofluorescent images of SOX17 (green) and Hoechst 33342 (blue)-stained nuclei were taken after 5 days and (B) the mean±s.e.m. (n>300) nuclear intensity of SOX17 was quantified. *P<0.05. Scale bar: 20 µm. a.u., arbitrary units.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 μg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 μM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay, Staining

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: Cell contractility is activated upon definitive endoderm induction. Mouse ESCs (CCE and R1) were grown in either definitive endoderm (DE) or pluripotency (PP) induction medium on decellularized fibroblast-derived ECM containing FRET-FN. (A) Confocal z-stacks of the FRET fibronectin matrix were captured after 0 (No Cells) and 5 days of induction and (B) the average FRET intensity ratio of the ESC associated matrix was quantified (n>16). ESCs were treated with 50 μM blebbistatin for 1 h at the end of definitive endoderm or pluripotency induction and (C) the average FRET intensity ratios of ESC-associated matrix (n>16) and (D) SOX17 gene expression were quantified (mean±s.e.m., n=3). For boxplots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 10–90th percentiles. *P<0.05.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 μg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 μM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay, Gene Expression

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: ECM signaling regulates definitive endoderm induction and contractility. (A) Mouse ESCs (CCE and R1) were grown in definitive endoderm (DE) or pluripotency (PP) induction medium on decellularized fibroblast-derived matrices supplemented with FRET fibronectin and either 0 μg/ml (– exogenous laminin) or 50 μg/ml (+ exogenous laminin) mouse laminin-111. Equal amounts of DOC-insoluble ECM were blotted for laminin or fibronectin after 1 and 5 days of ESC culture. (B) R1 ESC mean±s.e.m. (n>300) nuclear SOX17 staining intensity and (C) average extracellular matrix FRET intensity ratios (the box represents the 25–75th percentiles, and the median is indicated; the whiskers show the 10–90th percentiles, n>10) were measured after 5 days of definitive endoderm induction in the presence or absence of the α5β1-integrin function-blocking antibody BIIG2. *P<0.05. a.u., arbitrary units.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 μg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 μM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay, Staining, Blocking Assay

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: Laminin effects on contractility are specific to definitive endoderm. After 5 days in either definitive endoderm (DE) or pluripotency (PP) induction medium, and the average FRET intensity ratios of ESC-associated matrix were quantified (the box represents the 25–75th percentiles, and the median is indicated; the whiskers show the 10–90th percentiles) for mouse ESCs (CCE and R1) grown on decellularized fibroblast-derived ECM containing FRET-FN and either 0 μg/ml (– laminin) or 50 μg/ml (+ laminin) exogenous laminin. n>16. *P<0.05.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 μg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 μM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: Traction forces are necessary for definitive endoderm specification. Mouse ESCs (CCE and R1) were grown on decellularized fibroblast-derived ECM in definitive endoderm induction medium, with or without 10 μM blebbistatin. (A) Immunofluorescent images of SOX17 (green) and Hoechst 33342 (blue)-stained nuclei were taken after 5 days and (B) the mean±s.e.m. (n>300) nuclear intensity of SOX17 was quantified. *P<0.05. Scale bar: 20 µm. a.u., arbitrary units.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 µM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay, Staining

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: Cell contractility is activated upon definitive endoderm induction. Mouse ESCs (CCE and R1) were grown in either definitive endoderm (DE) or pluripotency (PP) induction medium on decellularized fibroblast-derived ECM containing FRET-FN. (A) Confocal z-stacks of the FRET fibronectin matrix were captured after 0 (No Cells) and 5 days of induction and (B) the average FRET intensity ratio of the ESC associated matrix was quantified (n>16). ESCs were treated with 50 μM blebbistatin for 1 h at the end of definitive endoderm or pluripotency induction and (C) the average FRET intensity ratios of ESC-associated matrix (n>16) and (D) SOX17 gene expression were quantified (mean±s.e.m., n=3). For boxplots, the box represents the 25–75th percentiles, and the median is indicated. The whiskers show the 10–90th percentiles. *P<0.05.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 µM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay, Expressing

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: ECM signaling regulates definitive endoderm induction and contractility. (A) Mouse ESCs (CCE and R1) were grown in definitive endoderm (DE) or pluripotency (PP) induction medium on decellularized fibroblast-derived matrices supplemented with FRET fibronectin and either 0 μg/ml (– exogenous laminin) or 50 μg/ml (+ exogenous laminin) mouse laminin-111. Equal amounts of DOC-insoluble ECM were blotted for laminin or fibronectin after 1 and 5 days of ESC culture. (B) R1 ESC mean±s.e.m. (n>300) nuclear SOX17 staining intensity and (C) average extracellular matrix FRET intensity ratios (the box represents the 25–75th percentiles, and the median is indicated; the whiskers show the 10–90th percentiles, n>10) were measured after 5 days of definitive endoderm induction in the presence or absence of the α5β1-integrin function-blocking antibody BIIG2. *P<0.05. a.u., arbitrary units.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 µM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay, Staining, Blocking Assay

Journal: Journal of Cell Science

Article Title: Traction forces mediated by integrin signaling are necessary for definitive endoderm specification

doi: 10.1242/jcs.166157

Figure Lengend Snippet: Laminin effects on contractility are specific to definitive endoderm. After 5 days in either definitive endoderm (DE) or pluripotency (PP) induction medium, and the average FRET intensity ratios of ESC-associated matrix were quantified (the box represents the 25–75th percentiles, and the median is indicated; the whiskers show the 10–90th percentiles) for mouse ESCs (CCE and R1) grown on decellularized fibroblast-derived ECM containing FRET-FN and either 0 μg/ml (– laminin) or 50 μg/ml (+ laminin) exogenous laminin. n>16. *P<0.05.

Article Snippet: Mouse ESCs [CCE cell line, Stem Cell Technologies ( Robertson et al., 1986 ); R1 cell line, American Type Culture Collection ( Nagy et al., 1993 )] were maintained on gelatin-coated substrates in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, 1 mM non-essential amino acids, 15% fetal bovine serum screened for mouse ESCs (Thermo Scientific), 100 µM 1-thioglycerol and 10 3 U/ml leukemia inhibitory factor.

Techniques: Derivative Assay