Metallic nanoparticles (NPs), as iron oxide NPs, accumulate in organs, cross the blood-brain barrier and placenta, and have the potential to elicit developmental neurotoxicity (DNT). Human stem cell-derived in vitro models may provide more realistic platforms to study NPs effects on neural cells, and to obtain relevant information on the potential for early or late DNT effects in humans. Primary neuronal-like cells (hNLCs) were generated from mesenchymal stem cells derived from human umbilical cord lining and the effects caused by magnetite (Fe(3)O(4)NPs, 1-50 mu g/mL) evaluated. Neuronal differentiation process was divided into stages: undifferentiated, early, mid- and fully-differentiated (from day-2 to 8 of induction) based on different neuronal markers and morphological changes over time. Reduction in neuronal differentiation induction after NP exposure was observed associated with NP uptake: beta-tubulin III (beta-Tub III), microtubule-associated protein 2 (MAP-2), enolase (NSE) and nestin were downregulated (10-40%), starting from 25 mu g/mL at the early stage. Effects were exacerbated at higher concentrations and persisted up to 8 days without cell morphology alterations. Adenosine triphosphate (ATP) and caspase-3/7 activity data indicated Fe(3)O(4)NPs-induced cell mortality in a concentration-dependent manner and increases of apoptosis: effects appeared early (from day-3), started at low concentrations (>= 5 mu g/mL) and persisted. This new human cell-based model allows different stages of hNLCs to be cultured, exposed to NPs/chemicals, and analyzed for different endpoints at early or later developmental stage.