Hazard assessment of methylmercury toxicity to neuronal induction in embryogenesis using human embryonic stem cells

Design of a high-throughput human neural crest cell migration assay to indicate potential developmental toxicants_suppl

“…Modern in vitro DNT assays are designed to model a specific biological key event (Leist et al, 2010;Crofton et al, 2011) at defined developmental stages (Stummann et al, 2009;van Dartel et al, 2009;Zimmer et al, 2011;van Thriel et al, 2012). For instance, assays have been developed that assess changes in early neural differentiation Pennings et al, 2012;Theunissen et al, 2012Theunissen et al, , 2013Krug et al, 2013b;Balmer et al, 2014;Waldmann et al, 2014;Rempel et al, 2015;Shinde et al, 2015), neurite outgrowth (Stiegler et al, 2011;Krug et al, 2013a), synaptogenesis (Harrill et al, 2011), gliogenesis (Fritsche et al, 2005) or myelination (Zurich et al, 2000(Zurich et al, , 2002.…”

Changes in miRNA Expression Profiling during Neuronal Differentiation and Methyl Mercury-Induced Toxicity in Human in Vitro Models

“…Undifferentiated human embryonic stem cells (H9, hESCs) (WiCell MOU 02-W177 SLA 10-W0409) were cultured and committed toward neural precursor cells for 10 DIV, followed up to full post-mitotic neuronal differentiation (28 DIV) as previously described [26]. Briefly, NIH-registered H9 cells were cultured at 37 °C and 5% CO 2 in maintenance medium (DMEM/F12 supplemented with 20% knock out serum replacement, 1% non-essential amino acids, 50 U/mL penicillin and 50 μg/mL streptomycin, 2 mM glutamine, 0.1 mM β-mercaptoethanol (all from Invitrogen, Carlsbad, CA, USA) and 8 ng/mL of human basic fibroblast growth factor (bFGF, Immunological Sciences, Rome, Italy).…”

“…The exposure to MeHgCl was performed during the early stages of the neuronal differentiation process (i.e., 1-36 days in vitro (DIV) for NT-2 cells and 1-10 DIV for H9 cells) since previous in vitro studies from our laboratory [19,20] and epidemiological evidence [21][22][23][24] indicate that, compared to the adult CNS, the early stages of brain development are more susceptible to toxicants. Moreover, the early exposure of neural progenitor cells to chemicals seems to be a more vulnerable time period than the later stages of neuronal differentiation [25,26]. Indeed, human embryonic stem cells (hESCs), at the stage of the neural precursor formation, have been shown to be affected by non-cytotoxic concentrations of MeHgCl [26,27].…”

“…Moreover, the early exposure of neural progenitor cells to chemicals seems to be a more vulnerable time period than the later stages of neuronal differentiation [25,26]. Indeed, human embryonic stem cells (hESCs), at the stage of the neural precursor formation, have been shown to be affected by non-cytotoxic concentrations of MeHgCl [26,27].…”

“…Furthermore, under specific culture conditions, the NT2-derived neurons can form functional synapses [33] and express a variety of synapse formation markers and neurotransmitters phenotypes [34]. Similarly, the H9 cell line can be committed toward neural progenitors [26] and further differentiated towards neuronal and glial phenotype [29,35].…”