PB1264 - Phenotypic heterogeneity within healthy donor endothelial colony forming cells explored by morphological analysis and RNA expression profiling

Tuesday, July 12, 2022

6:30 PM – 7:30 PM

Location: Exhibition

Presentation Format: In-Person

Presenting Author(s)

Sebastiaan N.J. Laan, MSc

PhD candidate
Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands
Leiden, Zuid-Holland, Netherlands

Co-Author(s)

RD

Richard J. Dirven

Research technician
Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands.
leiden, Zuid-Holland, Netherlands
Iris van Moort, PhD

Post Doc.
Department of Hematology – Erasmus MC, Erasmus University Medical Center - Netherlands
Rotterdam, Zuid-Holland, Netherlands
TK

Tom Kuipers

Lab technician
Sequencing Analysis Support Core, Department of Biomedical Data Sciences, Leiden University Medical Centre, Leiden, the Netherlands
Leiden, Zuid-Holland, Netherlands
HM

Hailiang Mei

Manager
Sequencing Analysis Support Core, Department of Biomedical Data Sciences, Leiden University Medical Centre, Leiden, the Netherlands
Leiden, Zuid-Holland, Netherlands
JE

Jeroen C.J. Eikenboom

Professor, Internist, Head of Div. T&H
Department of Internal Medicine, Division of Thrombosis and Hemostasis, Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands
Leiden, Zuid-Holland, Netherlands
Ruben Bierings, PhD

Principal investigator
Department of Hematology, Erasmus MC, University Medical Center Rotterdam, the Netherlands
Rotterdam, Zuid-Holland, Netherlands
SB

Suzan Boer

Postdoctoral Researcher
Department of Internal medicine, division of Thrombosis and Hemostasis, Leiden University Medical Centre, Leiden, the Netherlands
Leiden, Zuid-Holland, Netherlands

Background: Endothelial colony forming cells (ECFCs) derived from patients can be used to investigate pathogenic mechanisms of vascular diseases like von Willebrand disease in their native environment. Previously, considerable phenotypic heterogeneity has been observed between ECFC clones derived from healthy donors. This heterogeneity needs to be well understood in order to use the ECFCs as endothelial models for vascular disease.

Aims: Determine phenotypic and gene expression differences between ECFCs derived from healthy controls.

Methods: A total of 33 ECFC clones derived from 16 healthy controls were analyzed. Gene expression of a panel of Weibel-Palade body (WPB) content proteins, Von Willebrand Factor (VWF) transcription factors and endothelial cell markers was measured in all ECFC clones by qPCR. The transcriptome of a selection of ECFC clones (n=15) is currently being analyzed using bulk RNA sequencing as an unbiased method to examine the gene expression heterogeneity. Phenotypic profiling of the ECFCs was done using confocal microscopy, followed by automated WPB segmentation and morphometric analysis using CellProfiler. WPB quantity per cell, length and eccentricity were measured.

Results: Through hierarchical clustering of RNA expression profiles we could distinguish two major clusters within the ECFCs, which were mainly driven by differences in expression of endothelin-1, α-SMA, tPA, OPG, IL-8, VWF, GATA6, P-selectin and CD34 (Figure 1). Phenotypic profiling of these groups showed significantly more WPBs in cluster one compared to cluster two (Figure 2). Furthermore, the WPBs in cluster two were significantly shorter and rounder.

Conclusion(s): We observed a range of different RNA expression patterns between ECFC clones and clear differences in WPB count and structure, which can be used to more accurately classify ECFCs. The observed differences between the ECFC controls further emphasize the need to compare ECFC clones with similar characteristics when studying patient derived ECFCs.
Funding by SYMPHONY: NWO-NWA.1160.18.038 and Landsteiner Foundation for Blood Transfusion Research, Grant Number:1852.