Young Research Scientist Sharing Session

The µP-hPST Assay for
Developmental Toxicity
Screening andMechanism Study
Dr. Xing Jiangwa
Medical College,Qinghai University
Xining, Qinghai Province, China
2018.11.16

Environmental and Pharmaceutical Safety ofPregnant Women and Would-be Mothers
Antiemetic Thalidomide Tragedy（1960s）

Antineoplastics Side Effects

Radotinib (2016)

Imatinib (2015)

Zika Virus andMicrocephaly (2016)

Animal/Animal Cell Models for Teratogen Screening
Animals• Expensive• Time-consuming• Against 3R

Animal embryos and cells
Whole Embryo Culture (WEC)

Micromass (MM) AssayKistler & Howard, Methods in enzymology,1990

Yokoo et al., PNAS, 2005

• 40% inter-species
variations

Mouse Embryonic Stem Cell Test (mEST)

Zebrafish Developmental Assay

Hangingdrops

Beating
cardiomyocytes
Embryoid body
Seiler & Spielmann, Nature Protocol, 2011

Thalidomide tragedy

Padilla et al., Reproductive Toxicology, 2012

Current hPSC-Based Models• Current paradigm of assessing developmental toxicity using hPSCs
Gene Metabolites
expression

Gene Metabolites
expression

Drugs
TimeStem cells

Mesoendoderm induction

Kameoka et al., Toxicological Sciences, 2014

Diff / Non-Diffprocess

Tissue specificcells

Neural induction

Colleoni et al, Current MedicinalChemistry, 2012

Meganathan et al, PlosOne, 2012

Temporally controlled culture platforms may not reflect all chemicalinduced changes to developmental processes

An Ideal Representation of Embryonic Development
Change in structural motifs
during development
Motif = ∫space∫time [Differentiation][Migration]
Motif B

Motif A

(Cell migration)

Biochemical and mechanical gradients govern spatial patterning ofdifferentiation and morphogenesis.
(Rossant & Tam, Development, 2009； Martin & Wieschaus, Nature Cell Biology, 2010)

Our Approach to Engineer Human Development ModelEngineering environmental gradients in cell culture:
Biochemical gradient

Mechanical gradient•Micropatterning of multiple cells/matrices•Microfluidic patterning of soluble factors•Technical complexity•Inherent in geometrical cell population•Patterns differentiation in human adult stem
cells
AdipocytesToh et al., Integrative Biology, 2011

Kawada et al., Lab Chip, 2012

High

Low

Mechanical stressOsteoblasts
Ruiz et al., Cell Stem Cells, 2008

Our model
Human Pluripotent
stem cell (hPSC)
culture1. Spatially patterned
differentiation
2. Collective cellmigration

Developmental
structural motifMicropatterning & Mesoendoderm Induction
Generating micropatterned hPSC (µP-hPSC) colonies

Process of interest: Mesoendoderm differentiation
during gastrulation• Mesoendoderm induction factors:
Activin, FGF2 and BMP4Gilbert 6th edition

Patterned Mesoendoderm in µP-hPSC Colony•Mesoendoderm differentiation pattern corresponded withmechanical gradient
LowHigh
Mechanical stressMesoendoderm
markerBrachyuary (T)Mechanical gradient between colony edge and interior
F-actin

Cell-matrix adhesion Cell-cell adhesion
Integrin β1E-cadherin

Contractility
ppMLC0 hr

24 hrToh. Y.C., Xing, J., & Yu. H. Biomaterials 50 (2015): 87-97

Patterned Differentiation Coordinates Cell Migration•

Differentiation progression in spatially constrained µP-hPSC colony
Live-imaging over 3 days

200 µmCoordinated cell movement;
Displacement of differentiation
pattern
Collective cellmigration

Xing, J. et.al. Scientific Reports 5 (2015): 10038

X (space)
Y (time)

Differentiation
patterning atcolony edge

d1
d2
d3

Kymograph

Generation of Mesoendoderm Structural Motif
3D reconstruction of µP-hPSC
colony (day 3)

Invagination

ColonyInterior
http://w3.cns.org/university/pediatrics/Ch1Figure2a.jpg

Top of colony

Colony Edge

Mesoendoderm
Structural Motif

Xing, J. et.al. Scientific Reports 5 (2015): 10038

Bottom of colony

Developmental Toxicity Sensitivity
Penicillin GThalidomide• Antibotics, antibacterial drug
• Known non-teratogen

Untreated• Morning sickness (1960s); Multiple myeloma (now)• Inhibits angiogenesis, integrin adhesion signaling(Vargesson, BioEssays, 2009)+200 µg/ml Penicillin G+0.8 mM ThalidomideMorphologic Attributes Sensitive to Teratogens
Phase and fluorescentimages

Xing, J. et.al. Scientific Reports 5 (2015): 10038

Kymographs

Quantitative Morphometric Model of Teratogen Testing• Classification Rule:1) If LDC <= IC25H9 & IC25aHDF,Fluorescence imaging

the test compound is a teratogen;2) Otherwise: Non-teratogen.

TimagesMorphologic feature extraction

Feature clustering

Determine the lowest disruption
concentration (LDC)
Xing, J. et.al. Scientific Reports 5 (2015): 10038

No.

Compounds

US FDA
Pregnancy
Category*

1

Thalidomide

Pregnancy risk
category X

Teratogenic

N.A.

2

Retinoic acid(RA)Pregnancy risk
category D

Teratogenic

Teratogenic3Dpenicillamine

Pregnancy risk
category D

Teratogenic

Nonteratogenic

4

Valproic acid(VPA)

Pregnancy risk
category D

Teratogenic

Teratogenic

5

Penicillin G

Pregnancy risk
category B

Nonteratogenic

Nonteratogenic*: There are five categories.

In vivohuman data

In mEST

Teratogen Classification in The µP-hPSC Model
Our µP-hPSC model showed a better drug screening performance than mEST.

IC25H9Does
LDC<IC25H9?

In Our μPhPSC
Model

In Vivo
Human Data

In mEST

30 μM>1000 μM

Yes

Teratogenic

Teratogenic

N.A.

D0.36
ng/ml

> 2000ng/ml

Yes

TeratogenicTeratogenic

Teratogenic

DpenicillamineD

200μg/ml

278 μg/ml

Yes

Teratogenic

Teratogenic

Nonteratogenic

4VPAD

0.1 mM

0.13 mM

Yes

Teratogenic

Teratogenic

Teratogenic

5Penicillin GB

1000μg/ml

787 μg/ml

No

Nonteratogenic

Nonteratogenic

Nonteratogenic

Drugs

US FDA
Pregnancy
Category

LDC

1

Thalidomide

X

2

RA

3

No.

Xing, J. et.al. Scientific Reports 5 (2015): 10038

Assay Optimization and Validation——The µP-hPST• Two-step decision rule forteratogen classification.

• Linear regression results for Cmax
scale factor determination.

Test compound with known LDCacquired by morphometricquantification

1000
100

Step 1:10

Non-teratogen

LDC (µg/ml)

NoIs LDC <= IC25H9 &IC25aHDF?

10.1

Yes

No

Step 2:

0.01

y=7x
y=10x

Is LDC <= Scale factor of Cmax?

0.001

Non-Teratogen
Teratogen

Yes

TeratogenXing, J. et.al. Scientific Reports 7 (2017): 8491

0.00010.001

0.1Cmax (µg/ml)

101000The best concentration threshold is
7-10 fold of Cmax.

The µP-hPST Assay Performance• 30 compounds from various classes were tested in the µP-hPST.
• The assay accuracy is 97% with only one false negative compound
Diethylstilbestrol detected.
• Compounds which were misclassified in other assays but correctly
detected in the µP-hPST: Bosentan, Diphenylhadantoin, Furosemideand Lovastatin.
Table Summary of the uP-hPST assay performance
Assay

No. of compounds Sensitivity Specificity

Accuracy

uP-hPST
Targeted1Biomarker Assay

3093%

100%

97%

3679%

100%89%

92%

97%

94%

hPST271 (37 clearlylabelledcompounds)

1 Palmer, J. A.et.al. (2013). Birth Defects Res B Dev Reprod Toxicol 98(4): 343-363.
2 Kameoka, S., J. Babiarz, K. Kolaja and E. Chiao (2014) Toxicol Sci 137(1): 76-90.

Xing, J. et.al. Scientific Reports 7 (2017): 8491

SG and US Patents Granted

Application: To study the teratogenic mechanismsof tyrosine kinase inhibitors (TKIs)
Dextrocardia with situs• Women with cancer keep rising
inversus,hemivertebrae,• TKIs are antineoplastics withand cervical spina bifida
wide applications
Child with malformed
external ear (microtia)• However, it can cause various
birth defects and early
abortions.

Jain N, Sharma D, Agrawal R, et al. Medical Principles & Practice,2015, 24(3):291-293.
Yazdi M H, Faramarzi M A, Nikfar S, et al. Biomedicine &Pharmacotherapy, 2017, 95:1556-1564.

Proposed Workflow
Step 1: TKI Teratogenicity Assessment
Control+ TKI(Low)+ TKI(Medium)+ TKI(High)(1) Drug Dosing(2) Image and Data Processing(3) Teratogenicity Assessment
Define Drug Dose(DD)for Transcriptome Analysis

Step 2: Teratogenicity Mechanism Study(1) Transcriptome analysis

Control+ TKI 1

Reference sequencealignment
Gene expressionlevel(2) Downstream Validations

UTR analysis+ TKI 2+ TKI 3

SNP/InDel

KEGG enrichment

Antisense transcript
prediction

Gene structure
analysis
Gene differentialexpression

GO enrichment

Protein network

Expression patternclusteringsRNA analysis

Target/Biomarker Genes and Signaling NetworkProject Aim / Expected Outcome

Explore the key
molecular
mechanisms
leading to thedevelopmental
toxicity of TKIs.

Finding out thedominant signaling
pathways andpotential biomarkers
involved in TKI
teratogenicity.

Provide guidance
for antineoplastic
drug development/TKI drug
improvement.

Acknowledgments

Prof. Hanry Yu AP. Yi-Chin Toh(NUS)(NUS, IBN)

Dr. Shuoyu XuEmail: xingjiangwa66@163.com
Research Centre of Basic Medical Sciences,
Medical College, Qinghai University, China

Thank You!

Generation of Mesoendoderm Structural Motif

Mesoendoderm markers

Xing, J. et.al. Scientific Reports 5 (2015): 10038

EMT markers

Dose-dependent Testing Results•

Phase and fluorescent T images after 3-day drug dosing

Scale bar: 200 µm

Xing, J. et.al. Scientific Reports 5 (2015): 10038

Generation of Morphologic Clusters•

Unsupervised feature clustering

Xing, J. et.al. Scientific Reports 5 (2015): 10038

Detection of The Lowest Disruption Concentration (LDC)•Thalidomide*: p<0.0083 in post-hoc analysis.
Low: 30 μM ; Medium: 300 μM; High: 800 μM.

Xing, J. et.al. Scientific Reports 5 (2015): 10038

LDC: 30 µM