Journal: Molecular Medicine Reports

Article Title: Effect of cellular mass on chondrogenic differentiation during embryoid body formation

doi: 10.3892/mmr.2018.9272

Figure Lengend Snippet: Size of EBs and time (days) in the suspension culture affects the expression of germ layer specific genes. During suspension cell culture, the pluripotency markers, (A) NANOG and (B) SOX2, decreased among all studied variants. Gene expression analysis indicated that the mesoderm, (C) Brachyury and (D) MIXL1, germ layer was favoured in EBs formed from 500 and 1,000 cell wells when compared with the 1,500 and 2,000 cell wells at the end of suspension culture (day 15). The mesenchymal marker (E) SMA and endoderm marker (F) FOXA2 did not predominantly express in the studied variants. Gene expression analysis indicated that the ectoderm germ layer, (G) VIM and (H) PAX6, was favoured in EBs formed from 500 and 1,000 cell wells when compared with the 1,500 and 2,000 cell wells at the end of the suspension culture (day 15). The y-axis represents the relative expression of analysed genes, normalized to the BG01V cell line. Data are presented as the mean ± standard deviation. *P<0.05, as indicated. EBs, embryoid bodies; SOX2, sex determining region Y-box 2; MIXL1, mix paired-like homeobox; α-SMA, α-smooth muscle actin; FOXA2, forkhead box protein A2; VIM, vimentin; PAX6, paired box 6.

Article Snippet: The BG01V hESC line (American Type Culture Collection, Manassas, VA; USA) was cultured in mitomycin-C-treated mouse embryonic fibroblasts (MEFs; passage 3; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) seeded on a culture dish previously coated with MatrigelTM (Corning Incorporated, Corning, NY, USA).

Techniques: Suspension, Expressing, Cell Culture, Gene Expression, Marker, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Effect of cellular mass on chondrogenic differentiation during embryoid body formation

doi: 10.3892/mmr.2018.9272

Figure Lengend Snippet: Smaller EBs exhibit more prochondrogenic outcomes. Following collection of EBs from suspension culture, the 3 week chondrogenic differentiation protocol was performed and gene expression was analysed. Expression of (A) NANOG and (B) SOX2 decreased in all studied variants. Expression of (C) T, (D) SOX9 and (E) FGFR3 was significantly higher in EBs formed in 500-cell wells when compared with 2,000-cell wells. (F) CD44 was significantly expressed in differentiated 2,000-cell wells EB collected on the fifth day of suspension culture. (G) The low level of COL1A2 expression was observed in all studied variants following chondrogenic differentiation. (H) Expression of COL2A1 was significantly higher in EBs formed in 500-cell wells when compared with 2,000-cell wells. (I) ACAN expression increased only in EBs collected on day 5. Overall, 15 days of suspension culture resulted in a lower expression of analysed genes in chondrogenically differentiated EBs formed from 500- and 2,000-cell wells. The y-axis represents the relative expression of analysed genes, normalized to the BG01V or HC-402-05a cell line. Data are presented as the mean ± standard deviation. *P<0,05, as indicated. EBs, embryoid bodies; hESC, human embryonic stem cells; SOX, sex determining region Y-box; T, brachyury gene; FGFR3, fibroblast growth factor receptor 3; CD44, cluster of differentiation 44; COL, collagen; ACAN, aggrecan.

Article Snippet: The BG01V hESC line (American Type Culture Collection, Manassas, VA; USA) was cultured in mitomycin-C-treated mouse embryonic fibroblasts (MEFs; passage 3; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) seeded on a culture dish previously coated with MatrigelTM (Corning Incorporated, Corning, NY, USA).

Techniques: Suspension, Gene Expression, Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

doi: 10.1371/journal.pone.0091119

Figure Lengend Snippet: (A) TLR4 and NOD1 expression in hESC-EC (relative to expression in HUVEC) in vitro . Data are mean ± SEM (n = 3). Statistical significance was determined by one-sample t-test (*p<0.05) for NOD1 vs. TLR4 expression. (B) Representative immunocytochemistry images of hESC-EC (top) and HUVEC (bottom) stained for the NF-κB p65-subunit (red) in response to 1 hour treatment with or without, C12-iE-DAP (NOD1 agonist; 10 µg/ml), LPS (TLR4 agonist; 1 µg/ml) or IL-1β (1 ng/ml). Nuclei were stained with DAPI (blue; 5 µg/ml). Images were acquired using a Cellomics VTi HCS Arrayscanner with a CarlZeiss microscope. (C) LPS (TLR4 agonist; 1 µg/ml) and C12-iE-DAP (NOD1 agonist; 10 µg/ml) induced CXCL8 release after 24 hour stimulation. Data are mean ± SEM. For HUVEC, hESC-EC or BOEC, n = 4–8. For iPSC-EC, n = 2, single isolation. Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparison test for each cell type (*p<0.05) and by two-way ANOVA followed by Bonferroni's post-test for between cell types. Analysis was not performed on data from iPSC-EC.

Article Snippet: Experiments were carried out using the H7 hESC line provided under collaboration agreement with the Geron Corporation (Menlo Park, CA, USA) and with permission of the UK Stem Cell Bank.

Techniques: Expressing, In Vitro, Immunocytochemistry, Staining, Microscopy, Isolation, Comparison

Journal: PLoS ONE

Article Title: Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

doi: 10.1371/journal.pone.0091119

Figure Lengend Snippet: MSD analysis of cytokine (pg/ml) release from hESC-EC.

Article Snippet: Experiments were carried out using the H7 hESC line provided under collaboration agreement with the Geron Corporation (Menlo Park, CA, USA) and with permission of the UK Stem Cell Bank.

Techniques:

Journal: PLoS ONE

Article Title: Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

doi: 10.1371/journal.pone.0091119

Figure Lengend Snippet: TLR4 and NOD1 expression in (A) hESC-EC and (B) HUVEC before (pre-implant; open bars) and 21 days after (post-implant; filled bars) implantation in vivo (‘conditioning’). Data are mean ± SEM and are normalized at unity (1) to gene levels in pre-implant cells. HUVEC; NOD1 pre-implant n = 8, post implant n = 4: HUVEC; TLR4 pre-implant n = 10, post implant n = 3. hESC-ECs; NOD1 pre-implant n = 6, post implant n = 5: hESC-ECs; TLR4 pre-implant n = 10, post implant n = 6. Data was obtained from 2 independent experiments (using up to 12 rats per group). Statistical significance was determined by one-sample t-test where results were compared to a theoretical control of 1 (*p<0.05). ND = none detectable.

Article Snippet: Experiments were carried out using the H7 hESC line provided under collaboration agreement with the Geron Corporation (Menlo Park, CA, USA) and with permission of the UK Stem Cell Bank.

Techniques: Expressing, In Vivo, Control

Journal: PLoS ONE

Article Title: Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

doi: 10.1371/journal.pone.0091119

Figure Lengend Snippet: (A) Effect of LPS (1 µg/ml) or C12-iE-DAP (10 µg/ml) on CXCL8 release from hESC-EC and HUVEC after 24 hours. (B) Effect of Haemophilus influenzae (HIN) (10 5 –10 8 CFU/ml) on CXCL8 release from hESC-EC (solid line) or HUVEC (dashed line) after 24 hours. Data are mean ± SEM; n = 3 representative of 6 hESC-EC isolations. Statistical significance for responses to drugs or bacteria was determined by one-way ANOVA followed by Dunnett's multiple comparison test (p<0.05).

Article Snippet: Experiments were carried out using the H7 hESC line provided under collaboration agreement with the Geron Corporation (Menlo Park, CA, USA) and with permission of the UK Stem Cell Bank.

Techniques: Bacteria, Comparison

Journal: PLoS ONE

Article Title: Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

doi: 10.1371/journal.pone.0091119

Figure Lengend Snippet: (A) Relative expression (vs. GAPDH) of NOD1 following 48 hour incubation with NOD1 siRNA normalized to non-targeting siRNA; n = 6. (B) CXCL8 release from hESC-EC following 48 hour pre-incubation with non-targeting siRNA (open bars) or NOD1-siRNA (filled bars) and 24 hour treatment +/− C12-iE-DAP (10 µg/ml) or Haemophilus influenzae (HIN) (10 7 –10 8 CFU/ml); n = 7–8. (C) Effect of GSK'214 (300 nM; RIP2 inhibitor) or GSK'217 (300 nM; NOD1 inhibitor), given 30 minutes before a 24 hour treatment with HIN (10 7 CFU/ml) or C12-iE-DAP (10 µg/ml) on CXCL8 release; n = 4. It should be noted that GSK drugs increased CXCL8 release under basal conditions; for each experiment this was subtracted from treatment groups. For panel A, statistical significance was determined by one-sample t-test. For panel B statistical significance within siRNA groups was determined by one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05), and between groups by two-way ANOVA followed by Bonferroni's post-test (+p<0.05). For panel C statistical significance for the effects of inhibitor of C12-iE-DAP or HIN induced CXCL8 was determined by one-way ANOVA followed by Dunnett's multiple comparison test (*p<0.05).

Article Snippet: Experiments were carried out using the H7 hESC line provided under collaboration agreement with the Geron Corporation (Menlo Park, CA, USA) and with permission of the UK Stem Cell Bank.

Techniques: Expressing, Incubation, Comparison