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

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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

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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

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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

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• 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

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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

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• 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

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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

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• 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

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• 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

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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

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Transwell co-culture: hiPS-VSMC & HUVEC

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• 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

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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

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Acknowledgements

• Amy Pointon • Kelly Gray • Milka Budnik-Zawilska

• Cardiovascular in vitro Safety Team

• Andreas Ehlich• Matthias Gossmann• Tristan Pritchard-Meaker

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