Validation and Utility of Human iPS-Vascular Smooth Muscle Cells as an in vitro Vascular Model
Caroline Archer, Senior Scientist (Drug Safety and Metabolism) 30th November 2017
Background
• Drug-induced changes in blood pressure (BP) are a common and undesirable side-effect in drug development
Source: Laverty H, Benson C, Cartwright E, et al. British Journal of Pharmacology. 2011;163(4):675-693.2
Typical Vascular Safety Approach
In vitro screensMolecular: secP linked to haemodynamics
In vitro assays Wire-myography
In vivo studiesRat telemetry
Regulatory studiesDog telemetry; MIBP
Wire Myography Limitations• Tests the effect of compounds on the tone of resistance arteries
(mesenteric arteries)• Vasoconstrictive or vasodilatory effects may translate into blood
pressure changes
• Low throughput• Requires up to 3 rats per compound• Bespoke basis – flag required from secP screen• Some vessel to vessel variability • Species translatability
3
In vitro Vascular Model
• A cellular in vitro model is required, to allow detection and mitigation of these changes prior to in vivo studies
• Assess value of iPS-derived VSMC model; developed by Ncardia
• Healthy human fibroblasts à iPSC à vascular smooth muscle cells
• An alternative to primary human aortic smooth muscle cells
4Image Source: Zhao, Y., Vanhoutte, PM. and Leung, SWS. Journal of Pharmacological Sciences. 2015; 129(2):83-94.
Cellular in vitro Model
hiPS-VSMC Versus Primary Human VSMC
5
Feature hiPS-VSMC Primary VSMCLow Donor variability(Genetic and Environmental) ü û
Genetic manipulation ü û
Phenotype switching required û ü
Availability of cells Unlimited Limited Screening suitability ü û
Aims and Objectives
Develop and validate hiPS-VSMCs for potential future use as a 2D vascular model suitable for detection of vasoactive compounds
6
1Recapitulate data from Ncardia• Key vascular
smooth muscle cell markers (IF)
• Response to tool compounds (Ca2+ flux)
2Expand the set of vasoactive and non-vasoactive validation compounds
3Assess gene expression of key ion channels and receptors
4 5Develop a human 2D/3D co-culture with endothelial cells (HUVEC)
Compare alongside a primary human/rat model system
Expression of general VSMC surface markers on hiPS-VSMCs
7
• Positive expression of smooth muscle alpha-actin and calponin
• Cells could also be identified using RFP that was expressed following differentiation under the SM22 promoter
SM α-actin Calponin Merge TagRGF
Smooth Muscle alpha actin; a constituent of contractile apparatus Calponin; Ca2+ binding filament-associated protein, negative regulator of contraction SM22; earliest marker of differentiated smooth muscle cells, also present in proliferating SMC
Technology Platforms: Vascular Contractility Assay
8
FLIPR Tetra (Molecular Devices)
IonOptix(IonOptix)
• Calcium• High Throughput• Co-culture feasibility ü
• Calcium/Contractility• Low Throughput • Co-culture feasibility û
XCELLigence Cardio ECR(ACEA)
• Impedence• Med Throughput • Co-culture feasibility ü
Response to tool compounds
9
• FLIPR Tetra used to dose and measure Ca2+ flux in hiPS-VSMC
• Ca2+ flux induced in hiPS-VSMC by the tool compounds Ang II and ET-1
TagRGF
Initial characterisation confirmed the phenotype and pharmacological responses to tool compounds
Vasoactivity – wider validation compound set
• Vasocontriction/Vasodilation validation compounds were selected based:• Clinical compounds • Profile of known blood pressure change/vasoactivity in vivo
• Non-Vasoactive compounds:• Clinical compounds• No associated blood pressure change/vasoactivity in vivo
• 26 clinical compounds run through FLIPR Calcium assay • 15 Vasoconstrictors• 5 Vasodilators• 6 Non-Vasoactive
10
11
Compound In vivo Effect Result at Top Dose AZ1 Vasoconstrictor InactiveAZ2 Vasoconstrictor ActiveAZ3 Vasoconstrictor InactiveAZ4 Vasoconstrictor Inactive
Angiotensin II Vasoconstrictor ActiveEndothelin-1 Vasoconstrictor Active
AZ5 Vasoconstrictor InactiveAZ6 Vasoconstrictor ActiveAZ7 Vasoconstrictor InactiveAZ8 Vasoconstrictor Active
Ionomycin Vasoconstrictor ActiveAZ9 Vasoconstrictor Inactive
AZ10 Vasoconstrictor ActiveAZ11 Vasoconstrictor InactiveAZ12 Vasoconstrictor InactiveAZ13 Vasodilator InactiveAZ14 Vasodilator ActiveAZ15 Vasodilator InactiveAZ16 Vasodilator InactiveAZ17 Vasodilator ActiveAZ18 Negative Control InactiveAZ19 Negative Control InactiveAZ20 Negative Control InactiveAZ21 Negative Control InactiveAZ22 Negative Control InactiveAZ23 Negative Control Inactive
In vivo effect Active Inactive
Vasoconstrictors 7/15 8/15
Vasodilators 2/5 3/5
Negative control 0/6 6/6
Vasoactivity – Results
• Sensitivity– Vasoconstrictors, 47 %– Vasodilators, 40 %
• Specificity: – Vasoconstrictors, 53 %– Vasodilators, 60 %
Gene expression Analysis – Ion Channels
12
• A range of ion channels were expressed:
Channel Gene Expression Relative Expression Level
L-Type, Cav1.2 CACNA1C Low hAo>hVSMC>hiPS
T-Type, Cav3.1 CACNA1G Low hAo>hiPS>hVSMC
KATP KCNJ11 Low hAo>hiPS>hVSMC
KCa1.1 KCNMA1 High hAo>hiPS>hVSMC
Nav1.2 KCN2A Medium hAo>hVSMC>hiPS
Gene expression Analysis – Cell surface receptors
13
• A range of cell surface receptors with a known role in vasoconstriction or vasodilation were expressed:
• Difference in magnitude of expression observed – relevance unclear (the ‘right’ control?)
Receptor Gene Expression Relative Expression Level
ETA receptor EDNRA High hAo>hiPS>hVSMCThromboxane A2 receptor TBXA2R Medium hAo>hVSMC>hiPSAlpha- adrenoceptor ADRA2A Low hAo>hVSMC>hiPS
Summary:
1. Gene expression analysis & pharmacological validation
2. Need to consider the other cell types in the vasculature; co-culture
14
VSMC Co-culture
Can sensitivity and specificity of vascular model be improved?
• Increase physiological relevance by incorporating endothelial cells and/or
ECM into the model
1. 2D multicellular
2. 3D ECM
3. Transwell
15
Transwell co-culture: hiPS-VSMC & HUVEC
16
• hiPS-VSMC assay plate (≥7 days)
• Primary HUVEC on insert transwell (24 hours)
• hiPS-VSMC loaded with Fluo4
• Automated dosing
Treatment to Insert
hiPS-VSMCHUVEC
Transwell co-culture: hiPS-VSMC & HUVEC
17
hiPS-VSMC + HUVEC hiPS-VSMC
Vehicle1 µM ET-1
Vehicle1 µM ET-1
• Delayed Calcium flux
• HUVEC barrier/diffusion
Current challenges
Validation of hiPS-VSMC:
• Are they characteristic of a particular vascular bed? • Can the phenotype be matured i.e. expression of receptors? • Heterogenous population/purity to be determined
Development of in vitro vascular assay:
• Comparison with Primary human aortic smooth muscle cell model/rodent species
• Ca2+ transients as a surrogate for vasoactivity – high throughput assay for SMC contraction?
• Improve physiological relevance through development of a co-culture model
18
Acknowledgements
• Amy Pointon • Kelly Gray • Milka Budnik-Zawilska
• Cardiovascular in vitro Safety Team
• Andreas Ehlich• Matthias Gossmann• Tristan Pritchard-Meaker
19
Confidentiality Notice This file is private and may contain confidential and proprietary information. If you have received this file in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this file is not permitted and may be unlawful. AstraZeneca PLC, 1 Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0AA, UK, T: +44(0)203 749 5000, www.astrazeneca.com
20