Human Pluripotent Stem Cell Assay for Developmental ...

Defining the Reproducibility and Applicability Domain of devTOX quickPredict, a Human Pluripotent Stem Cell-

Based Developmental Toxicity Assay

Jessica Palmer

Associate Director of Toxicology

May 28, 2020

Stemina Biomarker Discovery

• History

– Established operations in 2007

– State-of-the-art mass spectrometry and

stem cell culture laboratories

• Expertise

– Human stem cell models (hES and iPS cells) of toxicity and disease

– Proprietary metabolomics platform

– Biology of Autism Spectrum Disorders

– Identification of biomarkers of toxicity and disease for diagnosis and more precise treatment, drug development, clinical study management, companion diagnostics

5/28/2020www.stemina.com 2

Stemina’s Approach


Human Pluripotent Stem Cell Biology

• Human pluripotent stem cells offer a unique opportunity to evaluate chemical toxicity using a human endpoint→ Eliminate species-specific differences in toxic response

• Differentiated cells function like their in vivo counterparts

• “Unlimited” supply• Applicable to high-throughput

systemsImage source: Estevan et al., (2011) Embryonic Stem Cells - Basic Biology to Bioengineering




LC-MS-based Metabolomics

Metabolome: Measure of Function



• Understand compound risk profiles faster

• Multiple exposure levels

• Minimal compound required

• Cost Savings


LC-MS-based Metabolomics

Stemina’sIn Vitro

Toxicology Assays

Stemina’s In Vitro Toxicology Assays

Exposure-based prediction of toxicity potential using targeted metabolomics and human pluripotent stem cells and differentiated cells


devTOXqP Development Overview

PluripotentSelf-renewal

• Initially developed using human embryonic stem cells– Optimized for use with human induced

pluripotent stem cells





• Exposed cells to a training set of 23 well-characterized compounds with known human developmental toxicity outcomes






• Exposed cells to a training set of 23 well-characterized compounds with known human developmental toxicity outcomes

• Measured changes in secreted and consumed metabolites using LC-MS-based metabolomics

• Identified two metabolites predictive of developmental toxicity potential– Ornithine & Cystine → o/c Ratio

Secretome



Ornithine and Cystine are Essential for Normal Embryonic Development

Ornithine

• Secreted by cells into the media

• Decreases or Increases with toxicity



Ornithine



• Urea cycle, Polyamine synthesis



Cystine

• Consumed by cells from the media

• Increases with toxicity

• Cystine/cysteine redox cycle →oxidative stress



Ornithine



• Urea cycle, Polyamine synthesis

Cystine

• Consumed by cells from the media

• Increases with toxicity

• Cystine/cysteine redox cycle →oxidative stress

Proliferation and Differentiation-Key processes for normal development


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devTOXqP Process

LC-MS Analysis

Spent Media

hPS CellCulture

Cells

Cell Viability

Test Article Exposure

Positive and Negative Controls included on each plate to ensure assay performance

hESC: WA09 (WiCell)iPSC: DYR0100 (ATCC)

5/28/2020

DataAnalysis

0 . 1 1 1 0 1 0 0 1 0 0 0

0 . 0

0 . 5

1 . 0

1 . 5

[ T e s t A r t i c l e ] , M

Ch

an

ge

in

Re

sp

on

se

(N

or

ma

liz

ed

to

Co

nt

ro

ls)

V i a b i l i t y

o / c R a t i o

d T P :

2 2 µ M

d T T

T P :

1 1 7 µ M

devTOXqP Predicts Potentially Toxic Exposures

Set using training set response @ human therapeutic Cmax to balance specificity and sensitivity.

• Toxicity is a function of exposure

• Identify exposure level that alters metabolism – Developmental Toxicity Threshold

(dTT)




(dTT)– Developmental Toxicity Potential

(dTP)– Compare to Cell Viability





(dTT)– Developmental Toxicity Potential

(dTP)– Compare to Cell Viability

• Determine non-toxic and toxicexposure windows



• No response in the o/c Ratio ratio and cell viability

→Little to no potential for developmental toxicity in vivo @ tested exposures

devTOXqP Interpretation



• o/c Ratio response independent of changes in cell viability• No change in cell viability



• o/c Ratio response independent of changes in cell viability• No change in cell viability

• Viability response occurs at a higher concentration (>3-fold)

→Potential to cause developmental toxicity in vivo @ ≥ dTP concentration


• Response for both the biomarker ratio and cell viability at a similar concentration (≤ 3-fold)

→Potential for developmental toxicity and/or embryo lethality in vivo @ ≥ dTP concentration



hESC and iPSC Accurately Predict the Potential for Developmental Toxicity

• Internal validation studies

• Diverse chemical set

– Pharmaceuticals, Agrichemicals, Cosmetics, Industrial, and Environmental chemicals

– Data compared to published human and in vivo results








ChemicalExpected

Pos/Neg

Stemina

iPSC

Prediction

Rat

NOEL/NOAEL

(mg/kg/day)

Rat

NOEL Cmax

(µM)

Rat

LOEL/LOAEL

(mg/kg/day)

Rat

LOEL Cmax

(µM)

Rat

Study Type

Rabbit

NOEL/NOAEL

(mg/kg/day)

Rabbit

NOEL Cmax

(µM)

Rabbit

LOEL/LOAEL

(mg/kg/day)

Rabbit

LOEL Cmax

(µM)

Rabbit

Study TypeSource

13-cis-Retinoic Acid 1 1 50 3.00 100 5.99oral dose on

GD103 3.16 15 10.32 GD8-11 Andrews et al. 2019; ICH S5(R3) Final

5-Fluorouracil 1 1 10 19.99 15 29.75single dose IP

GD9ND NA 40 853.32 SC GD12 Andrews et al. 2019; ICH S5(R3) Final

9-cis-Retinoic Acid 1 1 1.5 5 ND 0.5 Teixido et al., 2018

Abacavir 1 1 100 18.00 300 80.00 Oral (EFD) 350 700 Oral (EFD)Daston et al., 2014; Teixido et al.,

2018

Acebutolol 0 060

18NE

Oral (GD6-16)

IV (GD5-17)

60

2

NE

6

Oral (GD6-16)

IV (GD5-20)SECTRAL Product Monograph

Acycloguanosine

(Acyclovir)0 0 50

Maternal: 7.0604

Fetus: 8.659

NE

150

NA

133.659

sc (GD6-15)

sc GD9, 10, 1150

Maternal: 1.7318

Fetus: 1.421NE NA sc (GD6-18)

Moore et al., 1983 (PMID: 6662297)

Stahlmann et al., 1988 (PMID:

3190444)

• Rat and rabbit NOEL/LOEL data and corresponding Cmax values (when available) are compiled for all compounds tested.

• Working on a joint publication as part of a Tox21 Cross-Partner Project with NICEATM, EPA, FDA, NCAT that will describe these results and include IVIVE

Cell Type N Accuracy* Sensitivity Specificity PPV NPV

hESC 79 83% 90% 76% 81% 88%

iPSC 124 87% 88% 86% 88% 86%

*Accuracy reported is balanced accuracy (𝑆𝑒𝑛𝑠𝑖𝑡𝑖𝑣𝑖𝑡𝑦+𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐𝑖𝑡𝑦

2)







Higher Accuracy with devTOXqP vs. Other Model Systems for Human Developmental Toxicants

Model SystemNumber of

Compounds

devTOXqP

Concordance with

Model

Accuracy**

Model devTOXqP

Rodent* 35 74% 86% 89%

Rabbit* 28 79% 79% 86%

Mouse EST 23 65% 74% 91%

Zebrafish 24 75% 75% 92%

Whole Embryo Culture 26 69% 73% 96%

All values based on data from literature review of other model systems

*Current required in vivo tests **Assay accuracy compared to suspected human developmental toxicity potential

Palmer et al., (2013) Birth Defects Res B Dev Reprod Toxicol.


devTOXqP Reproducibility: Reference Compounds


Variability in Reference Treatment dTP Concentrations

Carbamazepine Methotrexate Thalidomide

dTP Mean (±SD), [µM] 1.4 (±0.7) 0.06 (±0.02) 0.16 (±0.07)

dTP Range [µM] 0.3 - 3.1 0.03 - 0.12 0.06-0.26

N 40 40 12

Data collected 2014-2020

Every new batch (thaw) of hPS cells is pre-qualified to check metabolism and ensure assay reproducibility.

devTOXqP Reproducibility: Plate Controls

Repeatability of Assay Endpoints: Intra- and Inter-Plate

Coefficient of Variation (CV)

EndpointMean Intraplate %

CV

Interplate CV (%)

Reference Negative Positive

Cell Viability 3.7 0.0 4.1 9.0

o/c Ratio 5.4 2.3 7.3 37.9

Ornithine 2.8 1.3 5.0 11.3

Cystine 4.7 2.6 8.5 29.0

Mean CV 4.1 9.9

Note: Calculations are based on the reference control normalized values. Mean

Intraplate % CV is the average CV for the reference, negative, and positive controls for

the specified endpoint.


Data for 272 experimental plates analyzed from 2016-2019

Every plate contains positive and negative controls to ensure that hPS cell metabolism and the developmental

toxicity predictions are within assay specifications.

devTOXqP Is Highly Predictive Across a Wide Range of Industries/Chemical Classes and Birth Defect Types

Birth Defect Category N Accuracy

Cardiovascular 12 83%

Central Nervous System 23 83%

Craniofacial 26 88%

Limb 15 93%

Skeletal 32 78%

Urogenital 8 88%

Other (e.g., Eye, Gastrointestinal) 4 75%

Embryo/Fetal Death 15 87%

Growth Restriction 19 74%

Skeletal Variations 7 100%


Use # Positive # NegativeBalanced Accuracy

Sensitivity Specificity

Pharmaceuticals/Vitamins 39 26 90% 87% 92%

Agrochemicals 21 11 80% 86% 73%

Industrial Solvents/Additives/Byproducts

6 10 90% 100% 80%

Personal Care Products 3 8 94% 100% 88%

Food Additives/Contaminants 3 9 94% 100% 89%

devTOXqP Is Highly Predictive Across a Wide Range of Pharmacological Classes

Pharmacological Classification # Positive # Negative Balanced Accuracy Sensitivity Specificity

Channel Modulators 3 1 100% 100% 100%

DNA Modifiers 2 0 100% 100% N/A

Enzyme Modulators 7 6 85% 86% 83%

Kinase Modulator 4 0 100% 100% N/A

Nucleoside Modulator/Central Metabolite Inhibitor

6 0 100% 100% N/A

Receptor Modulator 4 10 75% 50% 100%

Second Messenger Modulator 0 1 100% N/A 100%

Transcription Modulator 6 1 100% 100% 100%

Other 7 7 86% 86% 100%

Calculations are based on iPSC devTOXqP results. Combination of ICH reference compounds and additional pharmaceuticals tested at Stemina. When possible, predictions were based on non-clinical exposure data.



• Metabolized by sphingosine kinase to the active metabolite, fingolimod-phosphate– hPSC do not express this kinase – Evaluated active metabolite (S-fingolimod-

phosphate)

• Sphingosine 1-phosphate receptor (S1PR) Inhibitor – hPSC express S1PR3 but not S1PR1, 4, & 5

• Based on devTOXqP data, active metabolite elicits a response at lower concentrations than parent, indicating effect of parent compound are non-receptor mediated.

Receptor Modulators


Workflow for the ToxCast STM Dataset Tested in WA09 hES Cells

Zurlinden et al., 2020. Tox Sci. 174(2):189-209

Major Study Findings

• 19% of 1065 chemicals were predicted to have developmental toxicity potential.

• Assay performance compared with in vivo animal models of human prenatal developmental toxicity: 79%–82% accuracy, ≥ 84% specificity, ≤ 67% sensitivity– Sensitivity improved as more stringent weights of evidence requirements

were applied to the animal studies (more confidence in animal studies)

• Comparison with biochemical targets in ToxCast revealed positive and negative associations with the STM response, providing insights into the mechanistic underpinnings of the targeted endpoint and its biological domain.


devTOXqP Applicability Domain: ToxCast Data Set• Mined statistical correlations with 337 enzymatic and

receptor signaling assays in the NVS data set.• Sensitive pathways can be inferred from functional

annotation of molecular initiating events (MIEs) in the STM-positive and STM-negative domains.– Sensitive: receptor tyrosine kinases (RTKs) and their

associated downstream kinases and phosphatases →regulate cell growth, differentiation, and survival

– Insensitive: Ca++ second messenger generation (G-protein coupled receptor [GPCR] signaling) and nuclear receptor-mediated gene expression (aside from NR3C1) →many of these receptors are not expressed in hPS cells

• Results are consistent with the devTOXqP

applicability domain defined using pharmacological class approach.


Zurlinden et al., 2020. Tox Sci. 174(2):189-209

Conclusions• devTOXqP is an accurate and selective assay for predicting human

developmental toxicity.• devTOXqP can reproducibly measure the dose-response over time with

multiple cell lines, consumable and reagent lots, and technicians.• devTOXqP identifies a wide range of mechanisms but may not be applicable in

all cases, such as when the targets not expressed in hPS cells.– When targets are not expressed, could be used to provide evidence of off-target

effects that may impact development

• What’s next? – Incorporate in vitro to in vivo extrapolation (IVIVE)– EPA working on machine learning approach develop predictive models with

devTOXqP assay and other in vitro data and in silico models


Acknowledgements– Alan Smith

– Michael Colwell

– Mike Ludwig

– DelRay Sugden

– Daniel Braas

– Fred Kirchner

– Bob Burrier (COO, VP R&D)

– Beth Donley (CEO)Contact Information:

Jessica PalmerAssociate Director of Toxicology(608) 204-0104 x [email protected]


mailto:[email protected]

Select Stemina Publications


Palmer, JA, et al. (2020). A Targeted Metabolomics-Based Assay Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Identifies Structural and Functional Cardiotoxicity Potential.

Zurlinden, TJ, et al. (2020). Profiling the ToxCast Library With a Pluripotent Human (H9) Stem Cell Line-Based Biomarker Assay for Developmental Toxicity.

Palmer, JA, et al. Reprod Toxicol. (2017). A human induced pluripotent stem cell-based in vitro assay predicts developmental toxicity through a retinoic acid receptor-mediated pathway for a series of related retinoid analogues.

Zhu, H, et. al. ALTEX. (2016). Supporting read-across using biological data.

Palmer, JA, et. al. Birth Defects Res B Dev Reprod Toxicol. (2013). Establishment and Assessment of a New Human Embryonic Stem Cell-Based Biomarker Assay for Developmental Toxicity Screening.

Kleinstreuer, NC, et. al. Toxicol Appl Pharmacol. (2011). Identifying Developmental Toxicity Pathways for a Subset of ToxCast Chemicals Using Human Embryonic Stem Cells and Metabolomics.

For a complete list visit our website: http://www.stemina.com/news-publications/journal-publications

https://academic.oup.com/toxsci/advance-article/doi/10.1093/toxsci/kfaa015/5732694?guestAccessKey=e6ddc5a0-270b-4132-b943-7b0d386220d4

https://academic.oup.com/toxsci/advance-article-abstract/doi/10.1093/toxsci/kfaa014/5740594?redirectedFrom=fulltext

http://www.sciencedirect.com/science/article/pii/S0890623817301326

http://www.stemina.com/publication-supporting-read-across-using-biological-data

http://www.stemina.com/establishment-and-assessment-of-a-new-human-embryonic-stem-cell-based-biomarker-assay-for-developmental-toxicity-screening-3

https://na29.salesforce.com/ui/core/activity/ActivityReminderPage?at=1449084694442&_CONFIRMATIONTOKEN=VmpFPSxNakF4TlMweE1pMHdOVlF4T1Rvek1Ub3dOeTQ0TnpaYSx2QkhSVXV4RWFRRlNXWkJMXzVvdDZnLFpXTmhaVEU1

http://www.stemina.com/identifying-developmental-toxicity-pathways-for-a-subset-of-toxcast-chemicals-using-human-embryonic-stem-cells-and-metabolomics

http://www.stemina.com/news-publications/journal-publications

Stemina’s In Vitro Toxicology Assays

• All human, quantitative endpoints

• Excellent predictivity (sensitivity and specificity)

• Reduce animal use

• Highly reproducible

• Low cost

• Rapid turn-around

• Require small amounts of compound (15mg)


• Predicts human cardiotoxicity with >85% accuracy• Identifies functional and structural cardiotoxicants with a single assay• Can be combined with other assays or endpoints for a comprehensive

understanding of a test article’s cardiotoxicity liability • Methods/Deliverables:

– Performed with human iPS cell-derived cardiomyocytes – Predictive biomarkers and cell viability are evaluated at 8 exposure levels – Assay predicts concentration where a test article has potential for

cardiotoxicity potential based on changes in cell viability and 4 metabolites (lactate, 2’-deoxycytidine, thymidine, and arachidonic acid)

Exposure-based prediction of cardiotoxicity potential using human iPSC-derived cardiomyocytes using targeted metabolomics


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