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

Abstract

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 (Fe3O4NPs, 1–50 μg/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: β-tubulin III (β-Tub III), microtubule-associated protein 2 (MAP-2), enolase (NSE) and nestin were downregulated (10–40%), starting from 25 μg/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 Fe3O4NPs-induced cell mortality in a concentration-dependent manner and increases of apoptosis: effects appeared early (from day-3), started at low concentrations (≥5 μg/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.

Environmental Nanoparticles Reach Human Fetal Brains

“…The detrimental NP effects upon brain morphogenesis and microvasculature development are of deep concern. The in vitro work of Coccini and colleagues [ 47 ] is relevant: NP uptake resulted in a reduction in neuronal differentiation with a downregulation of β-tubulin III, microtubule-associated protein 2, enolase, and nestin. A dose-related effect was recorded, and the gene downregulation persisted for up to 8 days without cell morphology alterations.…”

“…Placental, embryonic, and fetal toxicity are at the core of the adverse outcomes of nanoparticles. The vulnerability of the brain is key, essentially because there is no question that a number of chemicals, and certainly NPs, can interfere with the highly precise neurodevelopmental processes taking place in intrauterine life [ 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 24 , 25 , 26 , 27 , 45 , 46 , 47 , 48 , 91 , 96 ]. Of critical importance is the relationship between intrauterine toxic exposures and risk for major neurological, psychiatric, and cardiovascular diseases, a subject discussed for a number of years since the pioneering work of Barker [ 19 ] and 21 century researchers discussing fetal and perinatal programming and neuropsychiatric, metabolic, and cardiovascular diseases [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ].…”

“…Our working hypothesis posits that in women chronically exposed to PM 2.5 above the annual current US Environmental Protection Agency (USEPA) standard (12 µg/m 3 ) and high NP concentrations, placental NPs could be taken as evidence for fetal–organ transfer [ 39 , 40 , 44 ]. We argue that the early-brain NP identification associated with structural organelle neural and blood vessel changes will have detrimental effects upon early neural cells and NVU formation and integrity, ultimately altering immature astroglial cells and morphogenesis and being a key factor for aberrant protein formation [ 31 , 32 , 33 , 34 , 35 , 46 , 47 , 48 ]. Highly neurotoxic combustion and engineered NPs are reaching fetal organs in development and are setting the bases for neurodevelopmental and neurodegenerative lifelong consequences.…”

Human Umbilical Cord Mesenchymal Stem Cell–Based in vitro Model for Neurotoxicity Testing

“…Moreover, hNLCs were shown to be susceptible to NP exposure, evidenced by a decrease in the expression of the specific neuronal markers β‐Tub III, MAP‐2, and NSE, by using immunofluorescence staining (Fig. 9) (Coccini et al., 2020).…”

“…In this respect, we have set up a new 2D cell-based model, namely, primary neuronlike cells generated from human MSCs derived from umbilical cord lining membranes Figure 1 Flowchart depicting the protocols described here to obtain MSCs from human umbilical cord lining membrane (Basic Protocol 1) and to obtain human NLCs from transdifferentiation of MSCs (Basic Protocol 2) to be used for neurotoxicity testing. (Coccini, Pignatti, Spinillo, & De Simone, 2020;De Simone, Spinillo, Caloni, Gribaldo, & Coccini, 2020). These hNLCs, serving as a source of "healthy" cells (that are not immortalized or cancer-derived cell lines), can be used with a testing battery that allows evaluation of a range of parameters, such as mitochondrial function and membrane integrity, and enable protein expression profiling of the neuronal differentiation process.…”