Journal: Scientific Reports

Article Title: Defining Minimum Essential Factors to Derive Highly Pure Human Endothelial Cells from iPS/ES Cells in an Animal Substance-Free System

doi: 10.1038/srep09718

Figure Lengend Snippet: (A) Timeframe involved in the stepwise differentiation from iPS/ES cells into endothelial cells. The representative markers of each cell type were labeled beneath them. MI: mesoderm inducer; VM: vasculogenic mixture. (B) Morphologies (upper panels: phase-contrast images; lower panels: nuclear staining) of the iPS cells induced under various conditions. Conditions were: Control (basal medium plus 10 µM of Y27632); ESM (the TeSR-E8 plus 10 µM of Y-27632); CHIR (Control plus 3 µM of CHIR99021); ACTIVIN (Control plus 2 ng/ml of Activin A). iPS cells (10,000) were seeded per well of a 12-well plate in the conditions and assayed 48 hours later. Scale bar: 200 µm. Hoechst 33258 served as the nuclear stain. (C) and (D) The cells were induced as in B and harvested after 48 hours. The mRNA levels of CDH1, CLDN3, CDH2, VIM, ITGB1, FN1, ZEB1, SNAI2, TWIST1 (C), HAND1, T, KDR, and NANOG (D) in each group relative to the iPS cells (ESM) were assayed. GAPDH served as a loading control. (E) Fluorescence-activated cell sorting for PDGFR+ (left) and KDR+ (right) cells. iPS cells were induced as the CHIR+ACTIVIN group in B and stained for sorting 48 hours later. (F) The cells in the red boxes of E were assayed for the mRNA levels of PDGFRA, KDR, NANOG, T, and HAND1 relative to the iPS cells. GAPDH served as a loading control. (G) Representative flow cytometry analyses for PECAM1+ cells on day 5 when the mesoderm was induced under the conditions in B. The iPS cells were induced as in B for 48 hours and the media were replaced with the basal medium plus 20 ng/ml of mVEGF-A for additional 72 hours before flow cytometry. (H) Quantification (mean ± s.e.m., n = 3) of G for the number of PECAM1+ cells (red columns) and the number of total cells (red plus white) on day 5. Percentages (mean ± s.e.m., n = 3) above each bar represent the ratios of PECAM1+ cell number relative to the total cell number. * denotes a significant difference compared with the control.

Article Snippet: Human iPS cell line DF19-9-7T (WiCell) ; human ES cell line TW1 (Bioresource Collection and Research Center, Taiwan); human ES cell line Ch8 (National Engineering Research Center of Human Stem Cells, China); Activin A (Prospecbio); CHIR-99021 and Y-27632 (SelleckChem); mouse VEGF-A (Prospecbio); human VEGF-A (Prospecbio); VEGF-E (Prospecbio); VEGF-B (Prospecbio); basic FGF (ACROBiosystems); insulin from Saccharomyces cerevisiae (Sigma); transferrin from Oryza sativa (Invitria); anti-human PECAM1 (eBioscience); anti-human KDR and anti-human PDGFRA (BD Bioscience); Matrigel matrix (BD Bioscience); poly(vinyl alcohol) (Sigma 360627); Pluronic F-68 (Sigma); bovine albumin fraction V (Invitrogen).

Techniques: Labeling, Staining, Fluorescence, FACS, Flow Cytometry

Journal: Experimental and Therapeutic Medicine

Article Title: TWEAK promotes endothelial progenitor cell vasculogenesis to alleviate acute myocardial infarction via the Fn14-NF-κB signaling pathway

doi: 10.3892/etm.2018.6703

Figure Lengend Snippet: Phenotypic characterization of cultured EPCs. EPCs exhibited a change toward mesenchymal transformation following transplantation in AMI mice. (A) UEA-1 lectin binding (green) and DiI-acLDL molecular probe uptake (red) were evaluated in early and late EPCs to confirm culture using photomicrographs. (B) EPCs expressed CD34, KDR and CD146, but were negative for CD45 and CD133, as assessed using flow cytometry. (C) Immunofluorescent staining was performed with antibodies against CD31 and α-SMA to detect the differentiation of EPCs labeled with DiD in veins and arteries following transplantation in AMI mice. White triangles indicate the differentiated EPCs. EPCs, EPC, endothelial progenitor cells; AMI, acute myocardial infarction; UEA-1, ulex europaeus agglutinin-1; CD, cluster of differentiation; KDR, kinase domain receptor; SMA, smooth muscle actin.

Article Snippet: Matrigel Matrix, and rat anti-mouse antibodies against fluorescein isothiocyanate (FITC)-conjugated CD34 (cat. no. 553733), phycoerythrin (PE)-conjugated KDR (cat. no. 555308), PE-conjugated CD45 (cat. no. 561087), and PE-conjugated CD146 (cat. no. 562196) were purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Techniques: Cell Culture, Transformation Assay, Transplantation Assay, Binding Assay, Flow Cytometry, Staining, Labeling

Journal: Stem Cell Reports

Article Title: Prostaglandin E 2 Is Required for BMP4-Induced Mesoderm Differentiation of Human Embryonic Stem Cells

doi: 10.1016/j.stemcr.2018.01.024

Figure Lengend Snippet: De Novo Synthesis of PGE 2 Is Required for BMP4-Induced Mesoderm Specification of hESCs (A) Schematic of different induction methods. Indo, indomethacin. (B) qPCR analysis of mesodermal marker gene expression levels in hESCs with BMP4 induction plus DMSO (−Indo) or indomethacin (+Indo) for 48 hr. ∗∗ p < 0.01 compared with −Indo group. n ≥ 3 independent experiments. (C and D) Immunostaining (C) and western blotting (D) analysis for BRA in hESCs with BMP4 induction plus DMSO (−Indo) or indomethacin (+Indo) for 48 hr. Scale bars represent 50 μm. (E) hemato-vascular precursors (HV) marker gene expression levels in hESCs with BMP4 induction plus DMSO (−Indo) or indomethacin (+Indo) for 48 hr and hemato-vascular precursors induction (SFDM + 50 ng/mL VEGF + 50 ng/mL bFGF) for 4 days. ∗ p < 0.05, ∗∗ p < 0.01 compared with −Indo group. n = 3 independent experiments. (F and G) Flow cytometry analysis of the percentage of KDR + CD31 + cells after hemato-vascular induction. ∗ p < 0.05 compared with −Indo group. n = 3 independent experiments. (H) Neuroectoderm (NE) marker gene expression levels in hESCs with BMP4 induction plus DMSO (−Indo) or indomethacin (+Indo) for 48 hr. ∗ p < 0.05 compared with −Indo group. n = 3 independent experiments. (I) qPCR analysis of tissue-specific gene expression levels in different engrafts. ∗ p < 0.05, ∗∗ p < 0.01 compared with −Indo group. n = 3 independent experiments. Error bars indicate SD. See also Figure S2 .

Article Snippet: The antibodies used were as follows: KDR-PE (560494) was from BD Bioscience; CD31-APC (17-0319-42) was from eBioscience.

Techniques: Marker, Expressing, Immunostaining, Western Blot, Flow Cytometry