Xcelligence dp system

We used Method B NCCs for this assay. We dissociated day-10 iPSC-NCCs into single cells with Accutase (Innovative Cell Technologies Inc.). We used the xCELLigence-DP system (Roche) with CIM-Plate 16 to measure the migration index of each type of iPSC-NCCs. The upper plate of CIM-Plate 16 was coated with fibronectin (10 μg/ml in PBS), and 100,000 cells were added to each upper well. NC medium without human recombinant EGF and human recombinant FGF2 was added into each upper well, and NC medium without human recombinant EGF and human recombinant FGF2 containing 10% fetal bovine serum was added to each lower well. Cells that migrated from the upper to the lower well were automatically measured by the xCELLigence system. Eventually, aphidicolin (Sigma, Saint Louis, MO) was used at the concentration of 10 μg/ml. Data were analyzed by Tukey's multiple comparisons test after one-way ANOVA and Sidak’s multiple comparisons test after two-way repeated measure ANOVA using GraphPad Prism software version 7.0a (GraphPad Software).

Cell proliferation and migration of cells was analyzed by xCELLigence DP system (Roche) [48 (link), 49 (link)]. Migration and proliferation of cells were detected by measurement of impedance of gold electrodes, that refers to “Cell Index”. Migration assays were performed in CIM-plates^® according to the manufacturer’s instructions and monitored for 24 h. For analysis of migration after ATRA treatment, cells were pre-treated with ATRA (5 μM) / DMSO for two days before seeding for migration analysis under treatment conditions. Cell proliferation was monitored continuously in E-plates^® for 120 h according to the manufacturer’s instructions. All experiments were performed in triplicates with 4 replicates.

In vitro angiogenesis assays were performed to investigate endothelial cell network formation of capillary-like tubes and migration. For the Matrigel network formation assay, each well of a 12-well Falcon tissue culture plate was evenly coated with 150 µl Matrigel (BD Biosciences, Bedford, MA, USA). At 24 h post-transfection, 6 × 10⁴ HUVECs in complete EGM-2 media were seeded per well, in triplicate. Network formation was assessed after 16 h by photographing the matrices using an Olympus IX71 inverted light microscope and a DP70 digital camera (Olympus America, Center Valley, PA, USA). Three independent fields were acquired from each well and the morphological aspects of the tube network quantified using the angiogenesis analyzer plugin [51 (link)] for ImageJ [52 (link)]. Cell migration was monitored using CIM-Plate 16 devices and the xCELLigence DP system (Roche Diagnostics, Mannheim, Germany). In this system, 1.6 × 10⁴ HUVECs (either transfected with shYULINK, vector control, or untreated) in normal culture media without FBS were seeded into the upper chamber. This upper chamber was then placed on the lower part of a CIM-device containing complete EGM-2 growth media as an attractant. Cell migration was monitored over a period of up to 24 h, by measuring changes in the impedance signal of the underside of the CIM-plate membrane (Roche Diagnostics).

The xCELLigence DP system (Roche Diagnostics GmbH, Germany) was used for measurement of migration. This system utilizes specialized culture plates containing gold electrode arrays beneath the bottom of individual wells. Cellular contact with the electrode surfaces increases the impedance across these gold arrays. This impedance value is measured by the DP system and reported in the dimensionless unit of cell index. Cells (3.0×10⁵/well) were seeded in six-well plates. After 24 h cultivation, cells were transfected with siRNA for 24 h, serum starved for 24 h, followed by reseeding (4.0×10⁴ cells/well) in CIM-Plate 16 (Roche). The lower chamber contained either 10% FBS or 10% FBS with 10 nM gastrin as attractant. Cell migration was monitored every 15 min on a RTCA DP instrument for 24 h. Data analysis was carried out using RTCA Software 1.2 supplied with the instrument.

Cell migration was assessed using the xCELLigence^® DP system (Roche Diagnostics GmbH, Mannheim, Germany). In this assay, cells are added to a membrane surface in the upper part of a CIM 16 plate, and cells can migrate through the membrane to the lower wells as a response to a chemoattractant signal. Gold electrodes are attached underneath the membrane, and cells in contact with electrodes reduce the conductivity. The change is interpreted inversely as the Cell Index (CI). Here, 5.0 × 10⁴ cells/well were seeded in 0.5% FBS/DMEM in upper wells containing inhibitor or vehicle (DMSO). As a chemoattractant, 5% or 10% FBS/DMEM was added to lower wells for normal or positive controls, respectively, with 0.5% FBS used to control for increased background migration (negative controls) in 48 h experiments. The plate was scanned every 15 min for 24 h or 48 h. The plotting of the CI curves was carried out using RTCA Software 1.2 (https://www.aceabio.com/products/rtca-dp/#tab-software) supplied with the instrument. For relative quantification, all technical replicates of a treatment were normalized to the intra-experiment normal control. A two-tailed Student’s t-tests were performed to find significant differences between groups (p < 0.05).