Reproducible differentiation and characterization of neurons from mouse embryonic stem cells

Abstract

Investigation on the effects of disease-associated mutations on neurodevelopment is an essential approach to understand the molecular basis of neurological disorders and can be achieved by generating suitable animal models. However, some of the mutations preclude development of animal models, leaving cell-based models as the only options. Mouse embryonic stem cells (mESCs) are attractive because of the well-established technologies for introducing disease-associated mutations and the feasibility of investigating the abnormalities during different stages of neurogenesis. Importantly, such transgenic mESCs enable large-scale screening and identification of the most promising small molecules and/or drug candidates before undertaking expensive animal studies. Although neuronal differentiation from mESCs is one of the earliest methods to be developed, we observed that the published as well as publicly available methods did not yield neurons consistently. Here, we describe a 16-day differentiation protocol that consistently induced differentiation of mESCs into neurons. This step-wise protocol enables monitoring of the neuronal differentiation process at different stages as well as characterization using the markers for immature and mature neurons by using immunocytochemistry and quantitative real-time PCRs. Development of a method for differentiating mouse ES cells into neurons. Differentiating the mouse ES cells into embryoid bodies prior to induction of neuronal differentiation results in better neuron formation.

Nitro-Oleic Acid Inhibits Stemness Maintenance and Enhances Neural Differentiation of Mouse Embryonic Stem Cells via STAT3 Signaling

“…The total loss of STAT3 in differentiated EBs is associated with a decrease of neuronal precursor cells [ 11 ]. In contrast to the study of Foshay et al, our data showed that NO 2 -OA treatment of EBs results in a significant increase of neuronal markers Nestin and Tubb3 (selected by [ 29 ]), and the presence of neurites was observed (see the morphology study for the details [ 25 ]). This discrepancy in the literature could be explained by the fact that NO 2 -OA inhibits the STAT3 phosphorylation at tyrosine 705 residue.…”

“…The gene expression of both neuro-specific markers was significantly up-regulated in NO 2 -OA-treated cells compared to both control and OA treatment at day 15 ( Figure 4 F–G). Neurogenisis was also evaluated using morphology studies, neurites were clearly visible at NO 2 -OA-treated cells at day 14 ( Figure 4 H; Figure S5 A,B) [ 24 , 25 ].…”

The Parkinson’s disease variant rs356182 regulates neuronal differentiation independently from alpha-synuclein

“…One caveat to this proposed mechanism is the fact that neurons rely heavily on signaling feedback for complete differentiation. We found that low seeding density resulted in aberrant cell detachment, while others have shown that overcrowding of neuronal stem cell inhibits differentiation ( 29 ). As a differentiation-specific gene, SNCA expression would also be indirectly affected by seeding density.…”

circRNA Acbd6 promotes neural stem cell differentiation into cholinergic neurons via the miR-320-5p-Osbpl2 axis

“…S1A). To characterize the roles of circAcbd6 on NSC differentiation, we performed RT-qPCR and Western blot analysis of three neuronal markers, Map2, Neurod1, and ChAT (19)(20)(21). The results showed that Map2, Neurod1, and ChAT in LV-circAcbd6 group were upregulated significantly compared with LVnegative control (NC) group (Fig.…”