Development and application of an in vitro human neural crest cell - - PowerPoint PPT Presentation

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Development and application of an in vitro human neural crest cell - - PowerPoint PPT Presentation

Aug 15, 2022 280 likes •491 views

Development and application of an in vitro human neural crest cell migration assay Johanna Nyffeler, PhD student, University of Konstanz, Germany Outline 2 1. Introduction 2. Develop a NCC migration assay suitable for high throughput 3.

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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

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Outline

1. Introduction 2. Develop a NCC migration assay

suitable for high throughput

3. Application of the assay:

Screen of a compound library

2

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What are neural crest cells (NCCs)?

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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

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Consequences of disturbed NCC function: Neurocristopathies

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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

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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
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Assay Development

2.

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

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day -1 day 0 day 2

6.35 mm 2 mm

Calcein Calcein viability measure migration measure stopper

cytotoxic specific effect

  • n migration
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Assay setup with controls:

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endpoint-specific control known positive control

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

48 h 24 h

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Testing new compounds

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specific unspecific proliferation-inhibitor

new „hits“ identified proliferation as

confounding factor

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Proliferation in the 24 h setup

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compounds from group III

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Prediction Model

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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%

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Conclusion part ‚Assay setup‘

  • advantages of the cMINC assay:
  • experimenter-independent
  • software for automated image analysis
  • high reproducibility
  • medium to high throughput
  • broad set of compounds tested:

tool compounds, positive controls, negative controls, etc...

  • special focus on proliferation
  • preliminary prediction model

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Application: Screening

3.

Arch Toxicol. 2017, in press

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Procedure

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flame retardants 12 pesticides 17 drug-like compounds 15 polycyclic aromatic hydrocarbo ns 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)

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Screening

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26/75 potential positive compounds

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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

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Follow-up assays

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Transwell migration Manual cell tracking

 all compounds confirmed  but not identical results

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Conclusion part ‚Screening‘

  • assay suitable for a screening
  • new migration-inhibiting compounds detected

especially organochlorine and organophoshorous compounds

  • compounds confirmed in other migration assays

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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

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Marcel Leist Tanja Waldmann Christiaan Karreman

THANK YOU!

Xenia Dolde Heidrun Leisner Giorgia Pallocca Alice Krebs