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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