Journal: Cell Research

Article Title: Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells

doi: 10.1038/cr.2014.59

Figure Lengend Snippet: Establishment of a hESC reporter line for endothelial cell-specific lineage detection and the two modified protocols for endothelial differentiation. (A) A human VE-cadherin (VEC)-targeting construct. A 2.5-kb fragment of the human VE-cadherin promoter region , was placed upstream of a cDNA encoding EGFP in a lentiviral expression vector (VEC-EGFP). (B, C) Spontaneously differentiating VEC-EGFP hESCs co-expressed EGFP (green) and the pan-endothelial cell marker CD31 ( B ; red), or endogenous VEC ( C ; red). BF, brightfield; Nuc, nuclei. Scale bars, 100 μm. (D) The VEC-EGFP + cells could successfully form capillary-like tube structures on Matrigel. Top, brightfield; bottom, VEC-EGFP. Scale bars, 50 μm (insets) and 200 μm. (E, F) Schematic diagrams of the two differentiation protocols. In method A (E) , hESC-derived EBs were initially generated in suspension cultures with BMP4 (day 0-3; phase 1), and the EBs were transferred to adherent conditions on day 3 and cultured with VEGF-A (day 3-7; phase 2). In method B (F) , dissociated single hESCs were directly seeded on adherent conditions and stimulated with BMP4 (day 1-4; phase 1) in N2/B27 medium, and the medium was switched to StemPro-34 medium containing VEGF-A on day 4 (day 4-6; phase 2). FACS analyses were performed to sort a VEC-EGFP + CD31 + endothelial cell population on day 7 (method A; E ) or day 6 (method B; F ), and the sorted cells were expanded in endothelial cell conditions in phase 3 (∼day 14). We tested the efficacies of > 60 bioactive molecules for effects on endothelial differentiation when they were administered in phase 1 (illustrated as “X”) or phase 2 (illustrated as “Y”) in addition to the basic protocol consisting of BMP4 in phase 1 and VEGF-A in phase 2. (G) Representative FACS results detecting the VEC-EGFP + CD31 + ECs at the end of phase 2 when the effective molecules for endothelial differentiation were used. (H) The combination of BMP4/GSK-3βI in phase 1 and VEGF-A/DAPT in phase 2 (left) with adjunctive PLGF and HGF in phase 2 (right) synergistically promoted endothelial differentiation and could generate VEC + CD31 + ECs with ∼50% - 60% efficacy. (I, J) Averaged percent EC (I) and sorted EC number (J) at the end of phase 2 in each treatment combination. * P < 0.01 and ** P < 0.0001 vs Basic (BMP4+VEGF-A); *** P < 0.0001 vs Basic+DAPT or Basic+GSK-3βI. Error bars, SD ( n ≥ 3).

Article Snippet: Cryosections (8 μm thick) were subjected to double immunofluorescence staining using the following primary antibodies: anti-GFP (rabbit polyclonal, Molecular Probes); anti-CD31 (rat monoclonal antibody, BD Bioscience); and anti-mouse VEC (rat monoclonal antibody, R&D).

Techniques: Modification, Construct, Expressing, Plasmid Preparation, Marker, Derivative Assay, Generated, Cell Culture

Journal: Cell Research

Article Title: Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells

doi: 10.1038/cr.2014.59

Figure Lengend Snippet: Subpopulation analyses of VEC + CD31 + ECs for identifying an endothelial progenitor population. (A) Expression of KDR and CD34 in the VEC + CD31 + ECs on day 6 was evaluated by FACS. (B) VEC + CD31 + ECs on day 6 were divided into three subpopulations (CD34 + CD14 − , CD34 − CD14 − , and CD14 + ) for further characterization. (C) CD14 mRNA expression in CD14 + and CD14 − populations sorted by FACS was analyzed with RT-PCR. (D) In vitro tube formation on Matrigel. Left, brightfield; right, EGFP. Scale bars, 100 μm. (E) High magnification photographs of tube formation in D (CD34 + CD14 − ECs). Arrows indicate the lumens of the tubes formed by implanted CD34 + CD14 − ECs. Top, brightfield; bottom, EGFP. Scale bars, 50 μm. (F) Quantitative results of total tube area in the tube formation assay. HUCBC, human umbilical cord blood cell; HUVEC, human umbilical vein endothelial cell. * P < 0.01. (G) The cell proliferation (MTS) assay showing the significant difference among the three subpopulations. * P < 0.01. (H) Generated VEC + CD31 + EC numbers on day 14 derived from 1 × 10 4 cells of each EC subpopulation sorted on day 6. * P < 0.01. (I) Molecular profiles of the three EC subpopulations on day 6, non-EC (VEC − CD31 − ), HUCBC, HUVEC, and CD34 + CD14 − EC (EP)-derived cell 30 and 60 days after initial FACS sorting through several passages were analyzed using PCR arrays (SABiosciences), focusing on endothelial lineage-related genes. The clustergram image of array data shows differentially expressed genes among the populations. Red indicates increased expression, whereas green indicates decreased expression. (J) Quantitative comparisons of mRNA expression of the selected genes in the PCR array data in I among the populations. (1) non-EC (VEC − CD31 − ); (2) CD14 + EC; (3) CD34 − CD14 − EC; (4) CD34 + CD14 − EC (EP); (5) HUCBC; (6) HUVEC; (7 and 8) EP-derived cell 30 days and 60 days after sorting. * P < 0.05 and ** P < 0.01 vs non-ECs. † P < 0.01 vs (2) and (3). # P < 0.01 and ## P < 0.0001 vs (3) and (4). Error bars, SD ( n ≥ 3).

Article Snippet: Cryosections (8 μm thick) were subjected to double immunofluorescence staining using the following primary antibodies: anti-GFP (rabbit polyclonal, Molecular Probes); anti-CD31 (rat monoclonal antibody, BD Bioscience); and anti-mouse VEC (rat monoclonal antibody, R&D).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, In Vitro, Tube Formation Assay, MTS Assay, Generated, Derivative Assay

Journal: Cell Research

Article Title: Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells

doi: 10.1038/cr.2014.59

Figure Lengend Snippet: The CD34 + CD14 − subpopulation among VEC + CD31 + ECs represents an endothelial progenitor population, and Notch signaling controls their proliferation or further maturation. (A) Schematic of the single-cell culture experiment. On day 6 in the endothelial differentiation, the single cells of the three subpopulations (CD34 + CD14 − , CD34 − CD14 − , CD14 + ) among hESC-derived VEC + CD31 + cells were sorted onto 96 well plates (1 cell/well) and cultured under endothelial cell conditions for 14 days. (B, C) Percent of single cells dividing (B) , defined as undergoing ≥ 1 cell division, and the average number of cell progeny per well after 14 days (C) were measured in each EC subpopulation. * P < 0.0001 vs CD34 − CD14 − and CD14 + EC. (D) Representative photomicrographs of the single CD34 − CD14 − (top), CD34 + CD14 − (middle), and CD14 + EC (bottom) culture assay. Scale bars, 50 μm (day 0) and 100 μm (day 3, day 9 and day 14). Day 3, day 9 and day 14, brightfield; day 0 and insets of day 14, VEC-EGFP. (E) Expression of CD34 and KDR on day 14 in the cultured cells derived from the EPs (VEC + CD31 + CD34 + CD14 − ) sorted on day 6. After sorting, EPs were cultured with VEGF-A and analyzed with FACS on day 14. (F) Representative images of western blot analysis for active Notch1 (the Notch1 intracellular domain (N1ICD)) in nuclear extracts of the cultured cells treated with VEGF-A with or without rhDll4 or DAPT. (G) Quantitative results in F are shown. Bands were scanned and quantified with Image J. The signal intensity was normalized to β-actin expression. (−) indicates no molecules. * P < 0.05 vs (−), ** P < 0.01 vs (−) and VEGF-A, # P < 0.01 vs VEGF-A, and VEGF-A + rhDll4. (H) Diverse ratios of VEC + CD31 + CD34 + CD14 − cells among VEC + CD31 + cells on day 14. 1 × 10 4 VEC + CD31 + CD34 + CD14 − cells (EPs) were sorted on day 6 and cultured with VEGF-A with or without rhDll4 or DAPT in phase 3. Notably, combined treatment with VEGF-A and DAPT maintained a higher ratio of VEC + CD31 + CD34 + CD14 − cells among the cultured cells on day 14, whereas VEGF-A and rhDll4 decreased that ratio ( P < 0.01). (I) The differential EC (VEC + CD31 + ) and EP (CD34 + CD14 − EC) ratios on day 6 induced with treatment with VEGF-A with or without rhDll4 or DAPT in phase 2. Error bars, SD ( n ≥ 3).

Article Snippet: Cryosections (8 μm thick) were subjected to double immunofluorescence staining using the following primary antibodies: anti-GFP (rabbit polyclonal, Molecular Probes); anti-CD31 (rat monoclonal antibody, BD Bioscience); and anti-mouse VEC (rat monoclonal antibody, R&D).

Techniques: Cell Culture, Derivative Assay, Expressing, Western Blot

Journal: Cell Research

Article Title: Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells

doi: 10.1038/cr.2014.59

Figure Lengend Snippet: DAPT enhances proliferation rather than differentiation of the KDR + precursors. (A) Differentiation of KDR + mesodermal precursors into the three cardiovascular lineages: endothelial cells (CD31 + ), smooth muscle cells (PDGFRα + ) and cardiomyocytes (cTnT + ). KDR + precursors were harvested on day 4 and cultured with VEGF-A with or without rhDll4 or DAPT up to day 8 prior to FACS analyses. (B) Quantitative results of the PDGFRα- and cTnT-positive ratios at day 8 in each KDR + -derived population, differentially treated as in A . * P < 0.05 and ** P < 0.01. Error bars, SD ( n = 5). (C) Immunocytochemical images of KDR + precursor-derived smooth muscle cells (left) and cardiomyocytes (right). SM-MHC and cTnT signals are indicated as red. DAPT attenuated VEGF-A-induced differentiation of KDR + precursors into smooth muscle cells and cardiomyocytes (indicated as arrows, respectively). Green, VEC-EGFP; blue, nuclei. Insets, brightfield. Scale bars, 50 μm (cTnT) and 100 μm (SM-MHC). (D) Expression of KDR in the human fetal heart (9 weeks of gestation)-derived cardiac mesenchymal cell fractions, treated with or without DAPT, was evaluated with FACS. (E) Immunocytochemical images of KDR + precursors in the human fetal heart-derived cardiac mesenchymal cell fractions. DAPT increased the number of KDR + precursors (red, arrows). Blue, nuclei; top insets and bottom left, brightfield. Scale bars, 50 μm (bottom) and 100 μm (top).

Article Snippet: Cryosections (8 μm thick) were subjected to double immunofluorescence staining using the following primary antibodies: anti-GFP (rabbit polyclonal, Molecular Probes); anti-CD31 (rat monoclonal antibody, BD Bioscience); and anti-mouse VEC (rat monoclonal antibody, R&D).

Techniques: Cell Culture, Derivative Assay, Expressing

Journal: Cell Research

Article Title: Manipulation of a VEGF-Notch signaling circuit drives formation of functional vascular endothelial progenitors from human pluripotent stem cells

doi: 10.1038/cr.2014.59

Figure Lengend Snippet: Successful engraftment of generated hPSC-derived EPs to form functional vessels in vivo . (A) Schematic of our finalized approach for generation of hPSC-derived EPs (VEC + CD31 + CD34 + CD14 − ). This is comprised of phase 1 and 2 in only 6 days, and sequential application of the following molecules: BMP4/GSK-3βI in phase 1 and VEGF-A/DAPT in phase 2. (B) Comparison of cell numbers of hPSCs before differentiation, generated EPs (day 6), and VEC + CD31 + ECs (day 14) after phase 3 expansion using our differentiation protocol. (C) Matrigel (MG) plugs with hPSC-derived EPs subcutaneously transplanted into NOD/SCID mice. In vivo live images of capillary vessel formation generated by the hPSC-derived VEC-EGFP + EPs 2 weeks after transplantation. BF, bright field. Scale bars, 100 μm (bottom) and 200 μm (top). (D) The MG plug with hPSC-derived EPs were harvested 4 weeks after transplantation (top), fixed in 2% paraformaldehyde (bottom, left), and subjected to fluorescent whole-mount immunostaining. The bottom-right panel shows anastomosis (arrowhead) between the neovessel formed by transplanted EGFP + EPs and a host vessel of NOD/SCID mice expressing murine VE-cadherin (red). Scale bars, 500 μm (top) and 200 μm (bottom, right). (E) Fluorescent immunostaining of cryosections of the MG plug in D shows anastomoses (arrowheads) between the neovessel formed by transplanted EGFP + EPs and host capillary vessels of NOD/SCID mice expressing murine VE-cadherin (red). Scale bar, 20 μm. (F) The neovessels formed by transplanted EGFP + EPs in a Matrigel plug with functional anastomosis (arrowhead) to native vessels were labeled with lectin (GS-IB 4 , blue). Scale bar, 20 μm. (G) In vivo live images of EGFP + EP-derived capillary vessels 3 months after transplantation. Black arrowheads indicate host angiogenic sprouting capillary vessels, whereas white arrowheads indicate neovessels formed by transplanted EPs in connection with host sprouting vessels. Scale bars, 100 μm. (H) The transplanted cell numbers of EPs (VEC + CD31 + CD34 + CD14 − ) sorted on day 6, ECs (VEC + CD31 + ) sorted on day 14 or VEC-EGFP + HUVECs were positively correlated with quantitative total tube area in in vivo Matrigel plugs. EPs have a three- to fivefold efficacy of quantitative vascular tube formation in vivo , compared with ECs or HUVECs. * P < 0.05 and ** P < 0.01.

Article Snippet: Cryosections (8 μm thick) were subjected to double immunofluorescence staining using the following primary antibodies: anti-GFP (rabbit polyclonal, Molecular Probes); anti-CD31 (rat monoclonal antibody, BD Bioscience); and anti-mouse VEC (rat monoclonal antibody, R&D).

Techniques: Generated, Derivative Assay, Functional Assay, In Vivo, Transplantation Assay, Immunostaining, Expressing, Labeling

Journal: Frontiers in Microbiology

Article Title: Recombinant Human IFNα-2b Response Promotes Vaginal Epithelial Cells Defense against Candida albicans

doi: 10.3389/fmicb.2017.00697

Figure Lengend Snippet: Effect of rhIFNα-2b on VEC viability . VK2/E6E7 cells were treated with rhIFNα-2b for 24 h. The mean values for the remaining four values and standard deviations (error bars) are shown. ∗∗∗ , significant difference compared to 0 mg/mL of rhIFNα-2b control group ( P < 0.0001).

Article Snippet: Human VEC line, VK2/E6E7 cells (ATCC ® CRL-2616), were obtained from the American Type Culture Collection (Rockville, MD, USA) and grown cultured in K-SFM (Gibco, USA) supplemented with 5 ng/mL recombinant epidermal growth factor and 50 μg/mL bovine pituitary extract (Invitrogen Corporation, Grand Island, NY, USA) with 100 units/mL each of penicillin and streptomycin (Life Technologies, Grand Island, NY, USA) at 37°C with 5% CO 2 in a high humidity environment.

Techniques: Control

Journal: Frontiers in Microbiology

Article Title: Recombinant Human IFNα-2b Response Promotes Vaginal Epithelial Cells Defense against Candida albicans

doi: 10.3389/fmicb.2017.00697

Figure Lengend Snippet: Effect of rhIFNα-2b on the production of IL-2 (A) , IL-4 (B) , IL-6 (C) , IL-8 (D) , and IL-17 (E) (expressed as pg/mL) by the VEC line, VK2/E6E7 cells cultivated alone, grown with 1.25 mg/mL rhIFNα-2b, and infected with C. albicans (1 × 10 5 /mL). The supernatants were collected and the cytokine levels were assessed by performing an ELISA 12 h post-infection and a subsequent 24 h of co-incubation with 1.25 mg/mL of rhIFNα-2b. V represents the VECs cultivated alone; V+I represents VECs co-incubated with 1.25 mg/mL of rhIFNα-2b for 24 h; V+C represents VECs infected with Candida albicans for 12 h; V+C+I represents the VECs infected with C. albicans for 12 h, then treated with 1.25 mg/mL of rhIFNα-2b for another 24 h. ∗∗ , significant difference compared to the V group ( P < 0.001); ∗∗∗ , significant difference compared to the V group ( P < 0.0001).

Article Snippet: Human VEC line, VK2/E6E7 cells (ATCC ® CRL-2616), were obtained from the American Type Culture Collection (Rockville, MD, USA) and grown cultured in K-SFM (Gibco, USA) supplemented with 5 ng/mL recombinant epidermal growth factor and 50 μg/mL bovine pituitary extract (Invitrogen Corporation, Grand Island, NY, USA) with 100 units/mL each of penicillin and streptomycin (Life Technologies, Grand Island, NY, USA) at 37°C with 5% CO 2 in a high humidity environment.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Frontiers in Microbiology

Article Title: Recombinant Human IFNα-2b Response Promotes Vaginal Epithelial Cells Defense against Candida albicans

doi: 10.3389/fmicb.2017.00697

Figure Lengend Snippet: Effect of rhIFNα-2b on the production of vaginal epithelial-derived IgG (expressed in μg/mL) by the VEC line, VK2/E6E7 cells . Supernatants were collected, and the IgG levels were assessed by performing an ELISA after 12 h of infection and a subsequent 24 h of co-incubation with rhIFNα-2b. V represents the VECs cultivated alone; V+I represents the VECs co-incubated with 1.25 mg/mL of rhIFNα-2b for 24 h; V+C represents the VECs infected with C. albicans for 12 h; V+C+I represents the VECs infected with C. albicans for 12 h, then treated with 1.25 mg/mL of rhIFNα-2b for another 24 h. ∗∗∗ , significant difference compared to the V group ( P < 0.0001); ∗∗ , significant difference compared to the V+C group ( P = 0.004). Each sample was repeated three times. The error bars indicate the standard deviation.

Article Snippet: Human VEC line, VK2/E6E7 cells (ATCC ® CRL-2616), were obtained from the American Type Culture Collection (Rockville, MD, USA) and grown cultured in K-SFM (Gibco, USA) supplemented with 5 ng/mL recombinant epidermal growth factor and 50 μg/mL bovine pituitary extract (Invitrogen Corporation, Grand Island, NY, USA) with 100 units/mL each of penicillin and streptomycin (Life Technologies, Grand Island, NY, USA) at 37°C with 5% CO 2 in a high humidity environment.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Infection, Incubation, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: Recombinant Human IFNα-2b Response Promotes Vaginal Epithelial Cells Defense against Candida albicans

doi: 10.3389/fmicb.2017.00697

Figure Lengend Snippet: Effect of rhIFNα-2b on VEC-mediated anti-Candida activity . SEM of the control cells (A) , C. albicans infected cells at 12 h (B) , and rhIFNα-2b treated cells (C,D) . (C,D) represent the VECs infected with C. albicans for 12 h, then treated with 1.25 mg/mL rhIFNα-2b for another 24 h. Microvilli are indicated by small red arrows, filopodia are indicated with a small yellow arrow, pseudohyphae are indicated with a small blue arrow, and living VK2 cells indicated with white arrows.

Article Snippet: Human VEC line, VK2/E6E7 cells (ATCC ® CRL-2616), were obtained from the American Type Culture Collection (Rockville, MD, USA) and grown cultured in K-SFM (Gibco, USA) supplemented with 5 ng/mL recombinant epidermal growth factor and 50 μg/mL bovine pituitary extract (Invitrogen Corporation, Grand Island, NY, USA) with 100 units/mL each of penicillin and streptomycin (Life Technologies, Grand Island, NY, USA) at 37°C with 5% CO 2 in a high humidity environment.

Techniques: Activity Assay, Control, Infection