Abstract

Abstract

It is well recognized that angiogenesis, the
growth of new blood vessels from pre-existing vasculature, plays a fundamental
role in health and disease. For the discovery of new drugs targeting the vasculature,
it is common practice to use primary endothelial cells in culture systems but, a number of constraints impede their
routine application in drug discovery. This includes the limited supply and batch-to-batchvariations due to genetic
and other variabilities amongst donors. This introduces an inherent biological
variability component that is difficult to control and which may negatively impact assay reproducibility.

To meet the
demands of predictive preclinical vascular drug research, improved in vitro models
of the vasculature are required: assays that are amenable to high-throughput
screening, with a scalable and robust cell source, in a physiological relevant
cellular micro-environment. Human induced pluripotent stem cell (hiPSC)-derived cells of the cardiovascular system are promising candidates to fulfil
these requirements.

We have
developed a reproducible manufacturing process for the generation of large
numbers of iPSC-ECs (>90% CD31⁺) using a set of controlled
bioreactor systems and subjected the cells to a standardized microfluidic cell
culture platform (OrganoPlate) to develop an angiogenesis assay which is
compatible with high-content imaging and high-throughput screening.

Upon the
formation of vascular lumen in microfluidic channels, sprouting into a three
dimensional collagen-based matrix was triggered with an optimized gradient of
angiogenic factors. Total sprout  area,
total sprout length and migration distance of each sprout were quantified using
high content imaging. We demonstrate that, similar to primary endothelial cells,
iPSC-ECs reproduce important hallmarks of angiogenic sprouting, including the formation
of tip cells that display their characteristic filopodia and trailing stalk
cells that finally anastomose and form a perfusable lumen. Complete anastomosis
coincided with a significant decrease in permeability for a fluorescent tracer,
indicating maturation of the vessel structures. At the same time non-mature
angiogenic sprouts retracted and pruned.

Remarkably
we detected known anti-angiogenic compounds such as Sunitinib (a clinically
used VEGFR2 inhibitor) at an IC50 of 21 nM which is exactly in line with observations
in a target based assay. The transient glycolysis inhibitor 3PO, also known for
its anti-angiogenic activity similarly reduced the sprouting of hiPSC-ECs in a
concentration-dependent manner, suggesting that the process is VEGF-driven with
glycolysis as main energy source. The developed assay is robust (AW > 10)
and reproducible (Z-factor > 0.7), providing excellent opportunities for
predictive phenotypic compound screenings in drug discovery.

Altogether,
the combination of a standardized microfluidic cell culture platform with a
scalable and robust cell source is a major step in the standardization of
physiologically relevant in vitro angiogenesis assays, as it offers the
required robustness, compatibility and scalability to be integrated within drug
screening.