Functional characterization of iPSC-derived arterial- and venous-like endothelial cells

The current work reports the functional characterization of human induced pluripotent stem cells (iPSCs)- arterial and venous-like endothelial cells (ECs), derived in chemically defined conditions, either in monoculture or seeded in a scaffold with mechanical properties similar to blood vessels. iPSC-derived arterial- and venous-like endothelial cells were obtained in two steps: differentiation of iPSCs into endothelial precursor cells (CD31pos/KDRpos/VE-Cadmed/EphB2neg/COUP-TFneg) followed by their differentiation into arterial and venous-like ECs using a high and low vascular endothelial growth factor (VEGF) concentration. Cells were characterized at gene, protein and functional levels. Functionally, both arterial and venous-like iPSC-derived ECs responded to vasoactive agonists such as thrombin and prostaglandin E2 (PGE2), similar to somatic ECs; however, arterial-like iPSC-derived ECs produced higher nitric oxide (NO) and elongation to shear stress than venous-like iPSC-derived ECs. Both cells adhered, proliferated and prevented platelet activation when seeded in poly(caprolactone) scaffolds. Interestingly, both iPSC-derived ECs cultured in monoculture or in a scaffold showed a different inflammatory profile than somatic ECs. Although both somatic and iPSC-derived ECs responded to tumor necrosis factor-α (TNF-α) by an increase in the expression of intercellular adhesion molecule 1 (ICAM-1), only somatic ECs showed an upregulation in the expression of E-selectin or vascular cell adhesion molecule 1 (VCAM-1).

Flow cytometry analyses. hiPSCs-derived ECs, HUVECs and HUAECs were dissociated with non-enzymatic cell dissociation buffer (Gibco) for 10 min, followed by gentle pipetting and washes in PBS with 5% FBS. Single cells were aliquoted in PBS with 5% FBS (between 100,000 and 125,000 cells were used per condition) and stained with either isotype controls or antigen-specific fluorescent-conjugated antibodies for 30 min at 4ºC. For VE-Cadherin and vWF analysis cells were fixed with 1% paraformaldehyde (PFA) for 10 min, permeabilized with Triton 0.1% for 10 min, and were incubated with primary antibody for 1 h at room temperature followed by the secondary antibody for 30 min at room temperature. For ICAM-1 analysis cells were incubated with primary antibody for 1 hour at 4ºC, followed by the secondary antibody for 30 min at room temperature. The antibodies utilized and respective dilutions can be found in Supplementary  Table S1: List of antibodies used for immunofluorescence (ICC) and flow cytometry (FC) analyses . The flow cytometry analyses were performed in BD FACSCalibur and BD Accuri C6, data analysis was performed with FlowJo_V10. Ten thousand events were collected in each run. The percentages showed in the dotplots were calculated based on the isotype controls represented by light blue. Isotype controls had 1% overlap with the protein of interest.
Immunofluorescence analyses. Cells were fixed with 4% (v/v) paraformaldehyde (PFA) for 10 min. When necessary, cells were permeabilized with 0.1% (v/v) Triton in PBS (10 min) and blocked with 1% (w/v) BSA solution for 30 min at room temperature. Antibodies were diluted with antibody diluent background reducing solution (DAKO). Cells were incubated for 1 h at room temperature with the primary antibodies, washed with PBS (3x5 min washes), incubated with a secondary antibody for 30 min at room temperature and thereafter counterstained with DAPI. The antibodies used and respective dilutions are found in Supplementary Table S1. Immunofluorescence analyses were performed on a confocal LSM 710 microscope (Zeiss). Capillary-like networks formation on Matrigel. A 96 Multi well plate was coated with Matrigel (BD) (50 µL/well -Matrigel was thawed at 4ºC and both tips and plates were kept cold during the procedure) and placed at 37 ºC for 30 min. Cells were seeded on top of the polymerized Matrigel at a density of 4-10x10 3 cells/well in 100 µL of EGM-2 (Lonza) medium. After 6 h, bright field images were acquired in InCell Analyser HCA System, and capillary-like networks analyzed using ibidi ACAS image analysis.

Quantification of COUP-TFII positive cells in iPSC-derived
Dil-Ac-LDL Uptake. Dil-Ac-LDL (Harbor Bio-Products) was added to the cell culture medium (20µg/ml) and cells maintained at 37ºC for 4h. In the last 10 min of incubation, Hoechst dye (0.25µg/ml) was added to stain cel nuclei. Cells were then washed and maintained with medium. Images were acquired in the InCell Analyser HCA System and analyzed by the corresponding software.
Monocyte-EC adhesion assay. HUVECs, HUAECs and iPSCs derived-ECs were cultured in 24well plates until confluence. The cells were then treated with TNF-α (10 ng/ml, Peprotech) a proinflammatory cytokine, for 6 h. In some wells, cells were counted after trypsinization to obtain the average number of cells per well. After 6 h, cells were washed 3 times in culture medium and cocultured with monocytes at the ratio of 1:2 for 30 min. The human monocyte cell line THP1 was used. These cells were previously loaded with the fluorescent dye CFSE (Molecular Probes) according to manufacturer instructions. Briefly, THP1 (1✕10 6 cells/ml) were incubated with CFSE (5 µM) for 15 min at 37ºC and washed three times in culture medium to remove the unbound dye. After 30 min, cells were washed 3 times with PBS to remove unbound THP1 cells, nuclei were stained with Hoechst (Molecular Probes), and the images were acquired in the InCell Analyser HCA System (GE Healthcare) and analyzed by the corresponding software. For non-treated and TNF-α-treated cells the percentage of THP1 cells was calculated relative to the total number of nuclei counted for each field. Each condition was performed in triplicate and a minimum of 12 fields were acquired per each well; approximately 300 cells were counted per each field. Intracellular Ca2+ variation measurements. HUVECs, HUAECs and iPSC-derived ECs were incubated with a membrane permeable acetoxymethyl (AM, 10 μM) derivative FURA-2/AM (1 mM in DMSO, Molecular Probes) and 0.06% (w/v) Pluronic F-127 (Sigma) diluted in endothelial serum free medium (without antibiotics) (Life TechnologiesA volume of 50 µl/well was used and cells were placed in the incubator for 1 h. The medium was then replaced by the respective basal medium and cells were further incubated for 30 min. Cells were washed twice with 100 μL sodium salt solution (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM Glucose, 10 mM HEPES-Na+ pH 7.4). The buffer was then replaced again (100 μl/well) immediately prior to incubating or not with test compounds. Cells were incubated with thrombin (5 U/ml, Sigma, T7009) or prostaglandin E2 (PGE2, 200 ng/ml, Cayman Chemical, 14010). Fluorescence was measured at emission 510 nm using two alternating excitation wavelengths (340 nm and 380 nm) using the microplate fluorescence reader (Spectramax Gemini EM, Molecular Devices). Each well was read every 6 s.
Trans-endothelial electrical resistance (TEER) measurements. Cells were plated in polycarbonate membrane transwell® inserts, pore size 0.4 µm for 12 well culture plates (Corning, 3401). Culture inserts were coated previously to the seeding of the cells with Matrigel (BD, 354234) diluted with DMEM (1:48) for 1 h. Cells were seeded at a density of 0.8 ×10 5 by insert and were left to stabilize for 3 days, after which thrombin (5 U/ml, Sigma, T7009) or PGE2 (200 ng/ml, Cayman, 14010) were added. The resistance of the monolayer was measured by a Millicell ERS-2 Voltohmmeter (Merck Millipore) after the addition of the stimuli. An empty filter was used to determine the background resistance. Three separate filters were used for each condition (type of cell), and the mean resistance was calculated after background subtraction. TEER measurements were performed again 48 h after the end of the experiment to verify the maintenance/or stability of the measured resistance values for each monolayer evaluated.

Characterization of nanofilms: mechanical tests and atomic force microscopy (AFM)
analyses. The mechanical properties of the nanofilms was evaluated by measuring their strain in response to an applied unidirectional stress. An Instron 4464 mechanical testing system equipped with a ±10 N load cell was used. Traction tests were performed on ten samples for each group. The nanofilms were detached from the substrate, by immersing them in water, then gently fished up and allocated between two aluminium clamps. All specimens were pulled at a constant speed of 5 mm/min until sample failure was reached. Data were recorded at a frequency of 100 Hz. The stress was calculated as the ratio between the load and the cross-section area of a tensile specimen, while the strain was calculated as the ratio between its extension and its initial length. The Young's modulus for each tested sample was extracted from its stress/strain curve, according to a standard procedure 2 . Imaging was performed with a Veeco Innova scanning probe microscope (Veeco Instruments Inc., Santa Barbara, CA) in dry state, operating in tapping mode, with oxidesharpened silicon probes (RTESPA-CP, Veeco Instruments Inc.) at a resonant frequency of ∼300 kHz. Each sample was gently scratched at its surface and then scanned across the scratch edge, over a 50 μm ✕ 50 μm area, recording 128 ✕ 128 values per each image. The resulting scan data were elaborated using the Gwyddion SPM analysis tool (http://gwyddion.net). They were levelled with the facet level tool to remove sample tilt, then the film thickness was evaluated as the difference between the average heights of a region of interest (ROI) selected on the nanofilm surface and the average height of the ROI on the silicon wafer. The analysis tool also quantified the average surface roughness for each image. For thickness and roughness measurements, three different images were acquired for each sample and three independent samples were analyzed for each sample type.
Expression of ICAM-1, E-Selectin and VCAM-1 by flow cytometry. HUVECs, HUAECs and iPSCs derived-ECs were cultured in 24-well plates until confluence. The cells were then treated with TNF-α (10 ng/ml, Peprotech, 300 01A) for 24 h and finally harvested using a Tryple Select   Mean fluorescence intensity of ICAM1 marker, as evaluated by flow cytometry. Cells were exposed to TNFα (10 ng/ml) during 24 h. Results are average ± SEM (n=3). (b) ICAM1 mRNA fold increase in HUVECs, HUAECs, AELCs and VELCs after exposure to TNFα (10 ng/ml) during 6 h. In each sample, ICAM1 expression was initially normalised by GAPDH expression followed by the normalisation with ICAM1 expression in cells not exposed to TNFα. Results are average ± SEM (n=3).