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Identification of angiogenesis modulators through comparative in vitro studies
with primary and iPSC derived endothelial cells.
Beibei Cai, Shuyun Lily Feng, Kathleen Kordestani, Patrick M. McDonough, Jeffrey H. Price and Daniel Rines.
High Content
Screening Services
Vala Sciences, Inc., La Jolla, CA (e-mail: [email protected], website: www.valasciences.com)
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Abstract
Angiogenesis, or blood vessel formation, is central to many physiological
and pathological processes, including growth, development, and wound
healing. It is also a critical step in tumor growth and metastasis. Therefore,
screening for compounds that modulate angiogenesis is useful for research
and development of anti-cancer drugs. We have developed two in vitro
model systems for assessing compounds that modulate angiogenesis.
Primary endothelia cells and induced pluripotent stem cells (iPSC) derived
endothelial cells were both used to study the angiogenetic properties of
compounds. Here we compare primary endothelial cells against iPSC
derived endothelial cells in their ability to form tubules under growth
factor stimulation, and their response to inhibitors such as suramin; as well
as other advantages and disadvantages of each model for upscaling
possibilities, our goal was to utilize these platforms in large-scale screening
of compounds that affect angiogenesis. Fluorescent staining with CD31
antibody was used to visualize tubules and imaged on an automated
fluorescent microscope. A custom image analysis algorithm, developed in
house, was employed to quantify tubules for image analysis. Total tubule
length and mean tubule length were reported as primary measurement
outputs for angiogenesis progression. Additional information that may
relate to the mechanism of action for compounds such as the node count
and area, and branch points were also analyzed. Both of these model
systems enabled us to screen a set of training compounds, as well as a
library of EPA ToxCast compounds, from which we were able to identify
both angiogenesis promotors and inhibitors. Our in vitro assays integrated
physiologically relevant models with this high-content screening approach
and allow us to perform for large-scale screening of compounds that affect
angiogenesis.
Materials and Methods
Materials. IC200 automated HCS microscope, CyteSeerReader automated
microscope control software, and CyteSeer high content image analysis
software were from Vala Sciences (San Diego, CA, USA). Bravo automated
liquid handling platform, BenchCel microplate handler, and VWorks
automation control software were from Agilent Technologies (Santa Clara,
CA, USA). HUVEC cells, and primary human dermal fibroblast cells and
media were from Cell Applications (San Diego, CA, USA). iPSC derived
endothelial cells and media were from Cellular Dynamics (Madison, WI,
USA). 384 well plates were from Greiner Bio-One (Frickenhausen,
Germany). Control compounds were from Sigma Aldrich (St. Louis, MO,
USA). Anti-CD31 antibody was from santa crutz biotechnology. Cell culture.
Primary Human Umbilical Vein Endothelial Cells or iPS derived endothelial
cells were co-cultured with primary human dermal fibroblast cells at 1:3
ratio . Cells were maintained with media and supplement per
manufacturers instructions.
High content screening. Images were acquired on an IC200 automated HCS
microscope at the end of the assay.
Results
Results
Figure 1
Figure 3
A)
A)
B)
iPSC derived endothelial cells
HUVEC
B)
C)
HUVEC
IC50: 23.917µM
Fig. 1. A) Schematic overview of the angiogenesis assay. iPSC derived or primary human umbilical vein
cells were co-cultured with human dermal fibroblast cells at 1:3 ratio. Cells were cultured for 3 days
before compound treatment for additional 3 days, after which and cells were fixed and stained for nuclei
and CD31, and imaged on a high content microscopy workstation (Vala IC200). B) Representative images
of the wells that were treated with vehicle (0.1% DMSO) or inhibitory control (suramin).
IPSC
IC50: 30.148µM
Figure 2
A)
Raw image
Segmentation
B)
0.1% DMSO
Fig. 3. A) Screening for a compound library demonstrate the sensitivity of the assay to
compounds that decrease (left) and increase (right) angiogenesis. Red line represents mean
of vehicle control. B) Comparison of the effects of the same compounds on angiogenesis
with HUVEC (blue line) or iPSC derived endothelial cells (red line). C) IC50 of one hit
compound that produced very similar results on both assay platforms .
Conclusions
100uM
suramin
Fig. 2. A) CyteSeer image analysis software automatically quantifies angiogenesis. Tubule like structures were
segmented based on of CD31 staining (green mask). For image analysis, the tubule like structures that pass
certain criteria for intensity and length were scored for angiogenesis. Total tubule length was used as the primary
output feature for angiogenesis B) HUVEC cells and iPSC derived endothelial cells showed similar results when
treated with tubule formation inhibitor suramin.
Vala Sciences has successfully created high content screening assays to measure
the effects of test chemicals on angiogenesis. Both primary HUVEC cells and iPSC
derived cells models were able to identify modulators for angiogenesis. Our in
vitro assays allow us to perform large-scale screening of compounds that affect
angiogenesis.
Contact
For more information regarding the IC200, CyteSeer, or high content screening
services, please visit: www.valasciences.com or contact us directly at :
[email protected], or call us toll-free at (858) 742-8252