Neuron-Like Cells Generated from Human Umbilical Cord Lining-Derived Mesenchymal Stem Cells as a New In Vitro Model for Neuronal Toxicity Screening: Using Magnetite Nanoparticles as an Example

Abstract

The wide employment of iron nanoparticles in environmental and occupational settings underlines their potential to enter the brain. Human cell-based systems are recommended as relevant models to reduce uncertainty and to improve prediction of human toxicity. This study aimed at demonstrating the in vitro differentiation of the human umbilical cord lining-derived-mesenchymal stem cells (hCL-MSCs) into neuron-like cells (hNLCs) and the benefit of using them as an ideal primary cell source of human origin for the neuronal toxicity of Fe3O4NPs (magnetite-nanoparticles). Neuron-like phenotype was confirmed by: live morphology; Nissl body staining; protein expression of different neuronal-specific markers (immunofluorescent staining), at different maturation stages (i.e., day-3-early and day-8-full differentiated), namely β-tubulin III, MAP-2, enolase (NSE), glial protein, and almost no nestin and SOX-2 expression. Synaptic makers (SYN, GAP43, and PSD95) were also expressed. Fe3O4NPs determined a concentration- and time-dependent reduction of hNLCs viability (by ATP and the Trypan Blue test). Cell density decreased (20–50%) and apoptotic effects were detected at ≥10 μg/mL in both types of differentiated hNLCs. Three-day-differentiated hNLCs were more susceptible (toxicity appeared early and lasted for up to 48 h) than 8-day-differentiated cells (delayed effects). The study demonstrated that (i) hCL-MSCs easily differentiated into neuronal-like cells; (ii) the hNCLs susceptibility to Fe3O4NPs; and (iii) human primary cultures of neurons are new in vitro model for NP evaluation.

Developmental Neurotoxicity Screening for Nanoparticles Using Neuron-Like Cells of Human Umbilical Cord Mesenchymal Stem Cells: Example with Magnetite Nanoparticles

“…hNLCs (at day 3 and 8) were examined under an inverted microscopy. Physico-chemical Fe3O4NPs properties in mesenchymal stem cell neurogenic differentiation medium (Table 2) were performed as detailed in De Simone et al [30]. The parameters were evaluated up to 48 h since fresh neurogenic medium was changed every 48 h.…”

“…Data are expressed as the mean ± (S.D.) of three measurements (data adapted from De Simone et al [ 30 ]).…”

“…In our recent study, we have reported an established stem cell-based in vitro model system to study various aspects of neurotoxicity (NT) and DNT [30] and proved that human umbilical cord lining-derived stem cells, such as mesenchymal stem cells (hMSCs), when differentiated into neuronal-like cells (hNLCs) may serve as one of the classical tools for the NT and DNT studies.…”

“…Applying this system, the toxicity effects caused by acute exposure (24–48 h) to increasing magnetite nanoparticles (Fe 3 O 4 NPs) concentrations (10–100 μg/mL) have been demonstrated in our lab using hNLCs at two specific stages of differentiation (i.e., at day 3 and 8). Cell density decrease (20–50%) and apoptotic effects were detected at ≥10 μg/mL in both types of differentiated hNLCs, and remarkably the three-day-differentiated hNLCs appeared more vulnerable than 8-day-differentiated cells [ 30 ].…”

The Recent Progress in Nanotoxicology and Nanosafety from the Point of View of Both Toxicology and Ecotoxicology

“…Stem cells can offer the opportunity to model the reproducibility of rare phenotypes in vitro [ 20 ]. For example, neuronal toxicity of Fe 3 O 4 NPs has been demonstrated by using the in vitro differentiation of the human umbilical cord lining-derived-mesenchymal stem cells (hCL-MSCs) into neuron-like cells (hNLCs) as an ideal primary cell source of human origin [ 21 ]. The study demonstrated that hCL-MSCs can be easily differentiated into neuronal-like cells and the hNCLs are susceptible to Fe3O4NPs, thus, human primary cultures of neurons could be a new in vitro model for NPs evaluation [ 21 ].…”