Development and application of an in vitro

human neural crest cell migration assay

Johanna Nyffeler, PhD student, University of Konstanz, Germany

Outline

1. Introduction

2. Develop a NCC migration assay

suitable for high throughput

3. Application of the assay:

Screen of a compound library

2

What are neural crest cells (NCCs)? 3

Gammill et al., 2003

Knecht et al., 2002

Migration

Delamination from

the neural tube

Differentiation into several cell types: - enteric neurons

- sensory neurons

- cartillage & bone

- melanocytes

- …

Neural crest cells

Proliferation

Consequences of disturbed NCC function:

Neurocristopathies 4

Migration

Proliferation

Hirschsprung‘s disease: - enteric neurons missing

- genes: RET, EDN3

Treacher-Collins syndrome: - craniofacial malformations

retinoic acid

ethanol

triazole fungizides

cyclopamine

Establish a test system for

- screening

- mechanistic exploration

Generation of neural crest cells from human pluripotent stem cells

Giorgia Pallocca

Goal: „Ring Assay“

• low throughput - varying scratch widths

- manual image acquisition

5

Zimmer et al. 2012

MINC assay cMINC assay

• high throughput - experimenter-independent

- automated image aquisition

Assay Development 2.

ALTEX. 2017;34(1):75-94

Assay Principle 7

day -1 day 0 day 2

6.35 mm

2 mm

Calc

ein

C

alc

ein

viability

measure

migration

measure

stopper

cytotoxic

specific effect

on migration

Assay setup with controls: 8

endpoint-specific control known positive control

“24 h“ assay able to detect specific NCC migration-inhibition

48 h

24 h

Testing new compounds 9

specific unspecific proliferation-inhibitor

new „hits“ identified

proliferation as

confounding factor

Proliferation in the 24 h setup 10

compounds from group III

Prediction Model 11

EC90V/EC90M

taxol 24

CdCl2 8.3

PCB180 6.6

LiCl 4.8

AraC 3.9

CytoD 3.6

retinoic acid 3.1

As2O3 2.8

acrylamide 2.7

staurosporine 2.6

colchicine 2.1

aphidicolin 2.0

AgNO3 1.5

MG-132 1.5

triton X-100 1.4

L-homocysteine 0.98

EC90V/EC75M

taxol 4.79

CdCl2 4.60

PCB180 4.43

LiCl 2.31

retinoic acid 2.20

CytoD 2.13

As2O3 1.51

acrylamide 1.50

colchicine 1.37

staurosporine 1.11

triton X-100 1.06

AgNO3 1.05

L-homocysteine 0.70

MG-132 0.69

AraC 0.43

aphidicolin 0.23

Migration inhibition at

EC90V [%]

PCB180 91.0

retinoic acid 66.9

CdCl2 56.6

LiCl 55.3

CytoD 53.4

taxol 49.6

As2O3 40.9

colchicine 40.4

acrylamide 39.9

triton X-100 29.6

AgNO3 27.6

staurosporine 27.6

AraC 17.9

MG-132 16.9

aphidicolin 13.7

L-homocysteine 9.3

endpoint-specific control

positive control

unspecific compound

Viability ≥ 90% and migration < 75%

Conclusion part ‚Assay setup‘

• advantages of the cMINC assay:

o experimenter-independent

o software for automated image analysis

o high reproducibility

o medium to high throughput

• broad set of compounds tested: tool compounds, positive controls, negative controls, etc...

• special focus on proliferation

• preliminary prediction model

12

Application: Screening 3.

Arch Toxicol. 2017, in press

Procedure 14

flame retardants 12

pesticides 17

drug-like compounds 15

polycyclic aromatic

hydrocarbons 17

industrial chemicals 9

negative controls 5

Screening cMINC assay

single concentrations

viability > 85% AND

migration < 80%

NO

Hit confirmation testing

concentration-response curves

YES

viability > 90% AND

migration < 75% NO

YES

compound

is

„negative“

compound is

a

„positive hit“

Follow-up assays

„NTP80-list“ (75 compounds)

Screening

15

26/75 potential positive compounds

Hit Confirmation

• 23 of 26 compounds confirmed

• hits fall all into 3 classes: - 10 flame retardants

- 7 pesticides

- 6 drug-like compounds

• many halogenated compounds

i.e. organochlorines

16

Follow-up assays 17

Transwell migration Manual cell tracking

all compounds confirmed

but not identical results

Conclusion part ‚Screening‘

• assay suitable for a screening

• new migration-inhibiting compounds detected especially organochlorine and organophoshorous compounds

• compounds confirmed in other migration assays

18

Take home messages

1. NCCs are an embryonic cell type &

target of developmental (neuro)toxicants

2. NCC migration can be assessed in vitro

3. cMINC assay is promising to screen for D(N)T compounds

4. Be careful when setting up an assay!

- use positive and negative controls

- confounding factors (i.e. proliferation)

5. different assays test for different biological processes

19

Marcel Leist

Tanja Waldmann

Christiaan Karreman

THANK YOU!

Xenia Dolde

Heidrun Leisner

Giorgia Pallocca

Alice Krebs