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.

Uliana De Simone,Teresa Coccini,Laura Gribaldo,Francesca Caloni,Arsenio Spinillo

doi:10.3390/ijms21010271

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.

Highlights

Among the different types of engineered NPs nanoparticles (NPs), the superparamagnetic iron oxide nanoparticles (SPIONs) are particles formed by small crystals of iron oxide commonly called magnetite Fe3O4 or maghemite γ-Fe2O3
Results related to immunofluorescence of GFAP showed the green fluorescence signal in hCL-mesenchymal stem cells (MSCs) and an increase of the fluorescence in human neuron-like cells (hNLCs) at 3 days, while hNLCs at 8 days displayed a weak signal, confirming that hCL-MSCs were capable to differentiate into hNLCs and into astrocytes (Figure 4B)
The fluorescence of synaptic makers indicated that the hNLCs were able to express both the pre- and post-synaptic proteins such as SYN, PSD95, and GAP43 (Figure 5)

Summary

Introduction

Among the different types of engineered NPs nanoparticles (NPs), the superparamagnetic iron oxide nanoparticles (SPIONs) are particles formed by small crystals of iron oxide commonly called magnetite Fe3O4 or maghemite γ-Fe2O3. These SPIONs have gained a huge interest due to their use for several biomedical and clinical applications (e.g., MRI contrast agents, treatments for anemia, magnetic sensing probes, and drug delivery agent) [1,2]. SPIONs have been developed for use as environmental catalysts and for incorporation into thermoplastics nanocomposites due to their pigmented properties The latter include several market products such as car tires, paints, etc. Iron oxide particulates, both fine/micron- and ultra-fine/nano-sized, are generated during anthropogenic activities related to the iron and steel industries, as well as during the NP manufacturing process, where they may represent a significant portion of the circulating air becoming a source of potential hazard for at-risk workers [5]

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