Functional assays in human embryonic stem cell derived neural crest cells to detect neurodevelopmental toxicants

Abstract

Proper neural development consists of several tightly regulated processes including e.g. differentiation, neurite outgrowth and migration. The main migratory cell population during neural development, the neural crest cells (NCC), emerge from the neural tube and migrate throughout the body to give rise to multiple cell types including neural and non-neural cells. To study potentially adverse effects of different compounds on the migratory potential of neural precursors, we used human embryonic stem cells (hESC) differentiated into NCC. We were able to keep the cells in a neural crest progenitor state, which allows the expansion and freezing of the cells. Furthermore, the NCC could be differentiated into peripheral neurons and Schwann cells, which confirmed their functional potential, while marker expression indicated the expected phenotype. To study the migratory potential of these cells and the effects of compounds on the migration capacity, we used the classical scratch assay which has been used successfully in many studies. LIVE cell imaging experiments demonstrated that these cells repopulate the scratch via migration independent of cell division. We could furthermore show that compounds which inhibit actin polymerisation inhibit the migration of these cells at concentrations that do not affect general cell viability or cell division. In addition to the classical scratch assay we used actin-GFP transfected cells to investigate the effects of different compounds on the actin cytoskeleton in real time using LIVE cell imaging. We therefore believe that our human based in-vitro test system is a powerful tool to detect potential neurodevelopmental toxicants and to study their mode of action.