Anti human cd34 fitc

For quantification of EPC, fresh EDTA-anticoagulated whole-blood samples were collected. 110 μl of peripheral blood was immediately stained (30 minutes in room temperature) with 20 μl of anti-human CD34 FITC (BD Pharmingen) and 5 μl of anti-human CD309 (VEGFR-2) PE (BD Pharmingen) monoclonal antibodies. Afterwards samples were lysed with BD FACS Lysing Solution (BD Bioscences), washed twice with PBS and fixed with use of CellFix (BD Biosciences) according to manufacturer’s procedure. Cells were analyzed by FACS Calibur flow cytometer and CellQuest Software (both from BD Immunocytometry Systems). Positive events gates was set via FMO (fluorescence-minus-one) controls. Based on positive surface expression of CD34 EPC were characterized as CD34+ CD309+ (CD34+ VEGFR-2+) cells localized in mononuclear cells (MNCs) gates and shown as percentages of total MNCs.

The iPSC clones were picked onto matrigel-coated 6-well plates in mTeSR medium. The next day culture medium was changed to EGM-2 medium (Lonza) supplemented with 25 ng/mL BMP4 (R&D), 3 µmol/L CHIR99021 (Selleck), 3 µmol/L IWP2 (Selleck) and 4 ng/mL FGF2 (Joint Protein Central) for 3 days. The cells were then cultured in EGM-2 medium supplemented with 50 ng/mL VEGF (HumanZyme), 10 ng/mL IL6 (Peprotech) and 20 ng/mL FGF2 (Joint Protein Central) for another 3 days. VECs were stained with anti-human CD34-FITC (BD biosciences, 555821, 1:200), anti-human CD201-PE (BD Biosciences, 557950, 1:200), and anti-human CD144-APC (BD Biosciences 561567, 1:200) and then sorted by a flow cytometry (BD FACSAria IIIu). IgG-FITC (BD biosciences, 555748), IgG-PE (BD biosciences, 555749) and IgG-APC (BD Biosciences, 555751) were used as isotype controls (Yang et al., 2017 (link)).

Recombinant human LIGHT (rhLIGHT) was purchased from R&D Systems (Minneapolis, MN, USA). Anti-human CD90-fluorescein isothiocyanate (CD90-FITC), anti-human CD44-FITC, anti-human CD105-FITC, anti-human CD34-FITC, anti-human CD45-FITC, anti-human CD19-FITC, anti-LTβR-PE, and FITC Mouse immunoglobulin G (IgG)-isotype control antibodies were purchased from BD Biosciences (San Diego, CA, USA). The Mesenchymal Stem Cell Growth Medium BulletKit™ (MSCGM™) was obtained from Lonza (Basel, Switzerland). For western blotting, antibodies against albumin, SRY-box transcription factor 17 (SOX17), forkhead box A2 (FOXA2), cyclin dependent kinase 1 (CDK1), CDK6, cyclin B1, cyclin D3, B-cell lymphoma-extra large (Bcl-xl), inhibitor of κB-α (IκB-α), protein kinase B (AKT), phospho-AKT, signal transducer and activator of transcription 3 (STAT3), phospho-STAT3, STAT5, phospho-STAT5, Rabbit IgG-horseradish peroxidase (IgG-HRP), and Mouse IgG-HRP were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against cytokeratin-18 (CK-18), CK-19, cytochrome P450 family 1 subfamily A member 1 (CYP1A1), CYP1A2, CYP3A4, CDK2, CDK4, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, Dallas, TX, USA). All reagents were obtained from Sigma–Aldrich (St. Louis, MO, USA).