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. Application of the assay: Screen of a compound library
What are neural crest cells (NCCs)? 3 Migration Proliferation Neural crest cells Knecht et al., 2002 Differentiation into several cell types: - enteric neurons - sensory neurons Gammill et al., 2003 - cartillage & bone Delamination from - melanocytes - … the neural tube
Consequences of disturbed NCC function: Neurocristopathies 4 Hirschsprung‘s disease: - enteric neurons missing Migration - genes: RET, EDN3 triazole fungizides Treacher-Collins syndrome: retinoic acid Proliferation - craniofacial malformations ethanol cyclopamine Establish a test system for - screening - mechanistic exploration Generation of neural crest cells from human pluripotent stem cells Giorgia Pallocca
Goal: „Ring Assay“ 5 cMINC assay MINC assay Zimmer et al. 2012 • low throughput • high throughput - varying scratch widths - experimenter-independent - manual image acquisition - automated image aquisition
Assay Development 2. ALTEX. 2017;34(1):75-94
Assay Principle 7 stopper day -1 day 0 day 2 Calcein specific effect on migration cytotoxic 2 mm migration measure Calcein viability measure 6.35 mm
Assay setup with controls: 8 endpoint-specific control known positive control 48 h 24 h “24 h“ assay able to detect specific NCC migration-inhibition
Testing new compounds 9 unspecific specific proliferation-inhibitor new „ hits “ identified proliferation as confounding factor
Proliferation in the 24 h setup 10 compounds from group III
Prediction Model endpoint-specific control positive control unspecific compound 11 Migration inhibition at EC90 V /EC90 M EC90 V /EC75 M EC90 V [%] taxol 24 taxol 4.79 91.0 8.3 4.60 PCB180 CdCl 2 CdCl 2 retinoic acid 66.9 PCB180 6.6 PCB180 4.43 56.6 4.8 2.31 CdCl 2 LiCl LiCl 55.3 AraC 3.9 retinoic acid 2.20 LiCl 53.4 CytoD CytoD 3.6 CytoD 2.13 taxol 49.6 retinoic acid 3.1 As 2 O 3 1.51 As 2 O 3 2.8 acrylamide 1.50 As 2 O 3 40.9 colchicine 40.4 acrylamide 2.7 colchicine 1.37 acrylamide 39.9 staurosporine 2.6 staurosporine 1.11 colchicine 2.1 triton X-100 1.06 triton X-100 29.6 AgNO3 27.6 aphidicolin 2.0 AgNO3 1.05 staurosporine 27.6 AgNO3 1.5 L-homocysteine 0.70 MG-132 1.5 MG-132 0.69 AraC 17.9 MG-132 16.9 triton X-100 1.4 AraC 0.43 aphidicolin 13.7 L-homocysteine 0.98 aphidicolin 0.23 L-homocysteine 9.3 Viability ≥ 90% and migration < 75%
Conclusion part ‚Assay setup ‘ 12 • 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
Application: Screening 3. Arch Toxicol. 2017, in press
Procedure 14 „NTP80 - list“ (75 compounds) Screening cMINC assay single concentrations negative flame controls 5 retardants 12 viability > 85% NO industrial AND chemicals 9 migration < 80% YES Hit confirmation testing polycyclic pesticides concentration-response curves aromatic 17 compound hydrocarbo is ns 17 „negative“ viability > 90% drug-like AND compounds NO migration < 75% 15 YES compound is a „positive hit “ Follow-up assays
Screening 15 26/75 potential positive compounds
Hit Confirmation 16 • 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
Follow-up assays 17 Manual cell tracking Transwell migration all compounds confirmed but not identical results
Conclusion part ‚Screening‘ 18 • assay suitable for a screening • new migration-inhibiting compounds detected especially organochlorine and organophoshorous compounds • compounds confirmed in other migration assays
Take home messages 19 NCCs are an embryonic cell type & 1. target of developmental (neuro)toxicants NCC migration can be assessed in vitro 2. cMINC assay is promising to screen for D(N)T compounds 3. Be careful when setting up an assay! 4. - use positive and negative controls - confounding factors (i.e. proliferation) different assays test for different biological processes 5.
THANK YOU! Marcel Leist Xenia Dolde Tanja Waldmann Heidrun Leisner Christiaan Karreman Giorgia Pallocca Alice Krebs
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