Sensitivity of neural stem cell survival, differentiation and neurite outgrowth within 3D hydrogels to environmental heavy metals

Abstract

We investigated the sensitivity of embryonic murine neural stem cells exposed to 10 pM-10 μM concentrations of three heavy metals (Cd, Hg, Pb), continuously for 14 days within 3D collagen hydrogels. Critical endpoints for neurogenesis such as survival, differentiation and neurite outgrowth were assessed. Results suggest significant compromise in cell viability within the first four days at concentrations ≥10 nM, while lower concentrations induced a more delayed effect. Mercury and lead suppressed neural differentiation at as low as 10 pM concentration within 7 days, while all three metals inhibited neural and glial differentiation by day 14. Neurite outgrowth remained unaffected at lower cadmium or mercury concentrations (≤100 pM), but was completely repressed beyond day 1 at higher concentrations. Higher metal concentrations (≥100 pM) suppressed NSC differentiation to motor or dopaminergic neurons. Cytokines and chemokines released by NSCs, and the sub-cellular mechanisms by which metals induce damage to NSCs have been quantified and correlated to phenotypic data. The observed degree of toxicity in NSC cultures is in the order: lead>mercury>cadmium. Results point to the use of biomimetic 3D culture models to screen the toxic effects of heavy metals during developmental stages, and investigate their underlying mechanistic pathways.

Keywords: 3D cultures; Heavy metals; Mechanistic pathways; Neural stem cells; Neurotoxicity.

Figure 1

Respective dose-response curves calculated for embryonic rat NSCs exposed to broad ranges of cadmium chloride (A), mercury chloride (B), or lead acetate (C), over 1 - 14 days.

Figure 2

(A) Representative immunofluorescence images of NSC cultures at day 7, differentiating into neural (TUJ1 staining) and glial (GFAP or MBP staining) lineages, in the presence of 10 pM to 1 nM cadmium chloride. Metal-free cultures served as controls. Cultures were counterstained with DAPI for cell identification. Scale bar: 50 μm. Blue dots indicate nuclei, green dots indicate neuron (TUJ1), pink dots indicate astrocyte (GFAP), and white dots indicate oligodendrocyte (MBP). Quantification of cells in each lineage was performed on day 7 (B) and day 14 (C). * indicates p < 0.05, ‡ indicates p < 0.001, and no notation for p > 0.05, compared to controls.

Figure 3

Quantified data from immunofluorescence images of NSC cultures, at day 7 (A) and day 14 (B), differentiating into neural (TUJ1 staining) and glial (GFAP or MBP staining) lineages, in the presence of 10 pM to 1 nM mercury chloride. Similarly, data was quantified from immunofluorescence images of NSC cultures, at day 7 (C) and day 14 (D), differentiating into neural (TUJ1 staining) and glial lineages, in the presence of 10 pM to 1 nM lead acetate. Metal-free cultures served as controls. * indicates p < 0.05, † indicates p < 0.01, ‡ indicates p < 0.001, and no notation for p > 0.05, compared to controls.

Figure 4

(A) Representative immunofluorescence images of neurite outgrowth within NSC cultures, at 1, 7 and 14 day time points. Quantification of neurite outgrowth at each dosage of cadmium (B), mercury (C), and lead (D), performed at regular intervals over a 14-day culture. Cadmium chloride, mercury chloride and lead acetate were each added at 10 pM to 1 nM dosage. Metal-free cultures served as controls. Scale bar: 50 μm. * indicates p < 0.05, † indicates p < 0.01, ‡ indicates p < 0.001, and no notation for p > 0.05, compared to controls.

Figure 5

NSC differentiation into motor neuron lineage (HB9 staining), at day 7 and day 14, in the presence of cadmium chloride (A), mercury chloride (B), or lead acetate (C), at 10 pM to 1 nM dosages. Metal-free cultures served as controls at each time point. * indicates p < 0.05, † indicates p < 0.01, ‡ indicates p < 0.001, and no notation for p > 0.05, compared to controls.

Figure 6

(A) NSC differentiation into dopaminergic neuron lineage (TH staining), at day 7 and day 14, in the presence of cadmium chloride (A), mercury chloride (B), or lead acetate (C), at 10 pM to 1 nM. Metal-free cultures served as controls at each time point. * indicates p < 0.05, † indicates p < 0.01, ‡ indicates p < 0.001, and no notation for p > 0.05, compared to controls.

Figure 7

(A) Representative immunofluorescence images of NSC cultures processed with JC-1 mitochondrial membrane potential assay kit to identify cells (green) with disrupted mitochondria. Cell cultures were treated with lead, mercury or cadmium at 100 pM to 10 nM for 14 days. High density of healthy cells (red channel) could be seen within metal-free controls. Scale bar: 200 μm. (B) Quantification of cells with disrupted membranes within these cultures. * indicates p < 0.05, † indicates p < 0.01, ‡ indicates p < 0.001, and no notation for p > 0.05, compared to controls.

Figure 8

Quantification of mechanisms by which various concentrations (10 pM – 100 nM) of heavy metal ions were influencing NSC cultures, over the 14-day culture period. (A) The percentage of cells with a compromised membrane was quantified using SYTOX® Green assay. (B) The percentage of cells committed to an apoptotic pathway was quantified using a YOPRO® assay. (C) Hoechst 33342 staining was used to quantify the cells with damaged DNA. Outcomes from each assay were compared to that noted within controls (0 M). * indicates p < 0.05 vs. controls. Differences in data between various concentrations within the same metal type was deemed statistically significant for p < 0.05 and denoted by #.

Figure 9

Amounts of various cytokines/ chemokines within NSC cultures exposed to cadmium, mercury or lead were quantified using a multiplexing laser bead assay (Mouse Cytokine/Chemokine Array 31-Plex). Respective supernatants (100 μL/metal/ concentrations) pooled over the entire 14-day culture duration was analyzed and compared to metal-free controls. Metal dosage was varied between 0.01 – 100 nM. Panels A, C and E depict the analytes detected at relatively higher levels (≥ 5 pg/mL), while panels B, D and F show markers released at ≤ 5 pg/mL.