Fibronectin coated plate

Half of the MNCs were resuspended in the Endocult medium (Stem Cell Technologies, Vancouver, BC, Canada) supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco/Invitrogen, Carlsbad, CA, USA). The suspension was seeded into one well of 6-well fibronectin-coated plates (BD Biosciences) and incubated for two days at 37℃, 5% CO₂, and 95% humidity. Then, the non-adherent cells were collected and replated onto 24-well fibronectin-coated plates (BD Biosciences). After 5 days, the culture medium was removed and the plates were washed with phosphate buffered saline. To increase the accuracy, the tests were performed in duplicate and the colonies were counted by two independent expert technicians.

A3 mESC were plated at 10,000 cells/cm² on 50 ng/mL fibronectin-coated plates (BD Biosciences) and cultured in Stage 1 Differentiation Medium.24 (link) After 3 days, the cells were replated at 10,000 cells/cm² on 50 ng/mL fibronectin-coated plates and cultured in Stage 2 Differentiation Medium containing 70% alpha-MEM (Mediatech) and 30% DMEM (Invitrogen) plus 2x Nutridoma CS (Roche), 1x penicillin-streptomycin (Invitrogen), 1x nonessential amino acids (Invitrogen), 2 mM L-glutamine (Invitrogen), and 0.05 mM 2-mercaptoethanol (Calbiochem), 10 ng/mL basic Fibroblast Growth Factor (Sigma), and 10 ng/mL VEGF (R&D Systems). On days 5, 7, 10, 12, and 14 of total differentiation, the differentiating cells were removed using Cell Dissociation Buffer (Invitrogen) for 30–60 minutes and analyzed for Tie-2 GFP or α-SMA RFP expression on an Aria III Flow Cytometer (BD). Data analyses were performed using FlowJo software (Tree Star Inc.). FITC- and PE-labeled compensation beads were used for compensation of potentially double positive cells. Unstained and undifferentiated A3 mESC were used as a negative control and gated to <5% of the negative control. Fluorescent populations of cells were analyzed for statistical significance using one-way analysis of variance and a post hoc Tukey’s HSD test using the R programming language.

CACs were isolated from 14 ml heparinized peripheral blood collected from 13 type 2 diabetic patients and 10 age-matched healthy subjects. All patients gave their informed consent before participating in the study, which was approved by the INRCA Institutional Ethical Board. PBMCs were isolated by density-gradient centrifugation with Ficoll (Ficoll-Paque™ PLUS) within 2 h of collection. 5 × 10⁶ PBMCs were plated on 24-well fibronectin-coated plates (BD Biosciences) and maintained in endothelial basal medium (Lonza) supplemented with EGM SingleQuots and 20% foetal calf serum for 4 days. After 4 days in culture, non-adherent cells were removed by washing with PBS whereas adherent cells were lysed directly in the culture wells. The CAC phenotype was confirmed as described previously [37] (link).