Asymptomatic neurotoxicity of amyloid ß-peptides (Aß1-42 and Aß25-35) on mouse embryonic stem cell-derived neural cells.

One of the strategies in the establishment of in vitro oxidative stress models for neurodegenerative diseases, such as Alzheimer's disease (AD), is to induce neurotoxicity by amyloid beta (Aß) peptides in suitable neural cells. Presently, data on the neurotoxicity of Aß in neural cells differentiated from stem cells are limited. In this study, we attempted to induce oxidative stress in transgenic 46C mouse embryonic stem cell-derived neurons via treatment with Aß peptides (Aß1-42 and Aß25-35). 46C neural cells were generated by promoting the formation of multicellular aggregates, embryoid bodies in the absence of leukemia inhibitory factor, followed by the addition of all-trans retinoic acid as the neural inducer. Mature neuronal cells were exposed to different concentrations of Aß1-42 and Aß25-35 for 24 h. Morphological changes, cell viability, and intracellular reactive oxygen species (ROS) production were assessed. We found that 100 µM Aß1-42 and 50 µM Aß25-35 only promoted 40% and 10%, respectively, of cell injury and death in the 46C-derived neuronal cells. Interestingly, treatment with each of the Aß peptides resulted in a significant increase of intracellular ROS activity, as compared to untreated neurons. These findings indicate the potential of using neurons derived from stem cells and Aß peptides in generating oxidative stress for the establishment of an in vitro AD model that could be useful for drug screening and natural product studies.

MiR-3099 is Overexpressed in Differentiating 46c Mouse Embryonic Stem Cells upon Neural Induction.

BACKGROUND: MicroRNAs (miRNAs) have a crucial role in gene expression regulation and protein synthesis, especially in the central nervous system. In developing mouse embryos a novel miRNA, miR-3099, is highly expressed, particularly in the central nervous system. This study aims to determine the expression of miR-3099 during cellular differentiation of 46C mouse embryonic stem cells after neural induction with N2/B27 medium. METHODS: 46C mouse embryonic stem cells were subjected to neural induction with N2/B27 medium. At 0, 3, 7, 11, 17, and 22 days after neural induction, the cells were screened for various pluripotent, progenitor, and differentiating/differentiated cells markers by immunocytochemistry and reverse-transcriptase polymerase chain reaction (RT-PCR). Stem-loop pulse RT-PCR was performed to determine the expression of miR-3099 at all selected time points after neural induction. RESULTS: Our findings showed that after induction, mouse embryonic stem cells differentiated into heterogeneous pools of cells containing neurons, astrocytes, and oligodendrocytes. Mouse embryonic stem cells and neural progenitor/precursor cells were also present in culture up to day 22 as indicated by RT-PCR analysis. Elucidation of miR-3099 expression during in vitro neural induction revealed that this miRNA was expressed throughout the differentiation process of 46C mouse embryonic stem cells. miR-3099 was expressed at higher levels on day 11, 17, and 22 as compared to day 0, 3 and 7 after neural induction. CONCLUSION: The level of miR-3099 expression was higher in differentiated mouse embryonic stem cells after neural induction. This finding suggested that miR-3099 might play a role in regulating neural stem cell differentiation. However, further characterisation of miR-3099 in a better characterised or optimised differentiated neural stem cell culture would provide increased understanding of the cellular function and molecular targets of miR-3099, especially in neuron development.