1. Introduction

Data of manuscript Blood-perfused Vessels-on-Chips stimulated with patient plasma recapitulate endothelial activation and microthrombosis in COVID-19

Contact: H.J.Weener@utwente.nl; Andries.vandermeer@utwente.nl

This dataset contains the data for the manuscript "Blood-perfused Vessels-on-Chips stimulated with patient plasma recapitulate endothelial activation and microthrombosis in COVID-19", to be published. Within the data are multiple sets, saved in individual .csv files:

* clinical data from included patients and controls; including admission time in hospital, diagnostic measurements when first included, outcome of admission, and VTE status during admission

* Cytokine ELISAs of IL-6, IL-10, TNF-a, IFN-y, CXCL-10, measured in the derived plasma of hospitalized patients

* Raw reads of RNA sequencing of hiPSC derived endothelial cells, after cells were stimulated with diluted patient plasma overnight to induce changes in their phenotype

* Raw pathogen counts based on said RNA sequencing data, where non-human RNA reads are quantified per sample

* Platelet coverage data after hiPSC-ECs were stimulated with derived plasma on-chip, where the stimulated chips were perfused with human whole blood to recapitulate the thrombotic adverse events that occur in some COVID patients


2. Methodological information

Clinical data from included patients and controls
Clinical data from patients was collected with informed consent via a standardised protocol approaved by the Medical Ethical Committee of the Amsterdam UMC location AMC. 
COVID-19 patients were included if they met the following inclusion criteria: age ≥ 18 years, a suspected SARS-CoV-2 virus infection, requiring additional oxygen, CRP ≥ 50 mg/l, D-dimer ≥ 0.5 mg/l, and were able to provide written informed consent. Patients were excluded if their medical history included venous thromboembolism, hereditary or acquired thrombophilia, or use of anticoagulant medication such as Direct Oral Anticoagulants or Vitamin K antagonist.

Plasma cytokines
Plasma concentrations of IL-6, IL-10, TNF-α, IFN-γ, and CXCL-10 were determined in duplicate, using sandwich enzyme-linked immunosorbent assay (ELISA, BioLegend). Assays were performed according to the manufacturer’s protocol. 

RNA sequencing
HiPSC-ECs for RNA sequencing were seeded in a 6 well plate and kept in culture until a monolayer formed. Citrated blood plasma was recalcified with 5:1 (v/v) with recalcification buffer that contained 1 M HEPES (Thermo Fisher), 63.2 mM CaCl2 (Sigma), and 31.6 mM MgCl2 (Thermo Fisher) diluted in MilliQ. Recalcified plasma was then diluted 1:4 in EGM-2 and added to the hiPSC-ECs. After overnight stimulation, culture medium was removed and cells were treated with 350 µl of 10 mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Sigma). RNA was isolated and purified from the samples with the Nucleospin RNA-kit (Macherey-Nagel).
RNA-sequencing and whole-genome transcriptome data were generated by Novogene (United Kingdom) on the Illumina NovaSeq6000 platform, paired-end sequencing and read length of 150 bp with a sequencing depth of 20M raw reads per sample.

Platelet coverage data
Each plasma donor was checked for platelet aggregation three separate times. Per run, three microscopy images of blood-perfused Vessels-on-Chip were taken and analysed blind by two researchers using ImageJ. Analysis protocol started by increasing the brightness and contrast, using the automatic function within the program. Then, images were cropped by excluding the 50 µm area directly adjacent to the walls of the channels, to exclude edge effects that could result in non-uniform shear rates, leading to platelet aggregation. The manual threshold function was used with dark background, to create a black and white image where only CD41+ pixels were black (platelet coverage). The thresholded image was analysed with the Analyse Particle(s) function, to obtain a covered area percentage of CD41+ pixels.

3. Data specific information

Abbreviations
Seq_name: assigned name in RNA-sequencing data
CoV: COVID-19 positive patient
Pn: COVID-19 negative patient, hospitalized with pneumonia
Ctrl: Healthy control group
CRP: C-reactive protein
VTE: Venous thromboembolism
ICU: Administered to Intensive care unit
PE: Pulmonary embolism
N.A.: Not applicable measurement for said donor
IL-6: Interleukin-6
IL-10: Interleukin-10
TNF-a: Tumor necrosis factor alpha
IFN-y: Interferon gamma
CXCL10: C-X-C motif chemokine ligand 10

4. Sharing and Access information

This work is licensed under CC BY-NC 4.0. To view a copy of this license, visit https://creativecommons.org/licenses/by-nc/4.0/