Resumo

Objectives: Mouse embryonic stem cells (ESCs) can be differentiated in vitro into a variety of somatic cell types, including lineages from all three germ layers. However, protocols for inducing neural differentiation are typically time consuming multistep processes. Here, we have investigated whether 2,4-dinitrophenol (DNP), a neuroprotective compound shown to potentiate neural differentiation in a murine neuroblastoma cell line (Wasilewska-Sampaio et al., 2005), induces differentiation of embryonic stem cells into a neuronal phenotype. Methods: ESCs were allowed to aggregate into embryoid bodies (EBs) for 4 days.Treatment with DNP was compared to retinoic acid (RA), the most used morphogen to induce neural differentiation in ESCs. EBs were treated with 20 μM DNP, 5 μM RA or maintained in control conditions for 4 days. EBs were then fixed in 4% formaldehyde, embedded in Tissue Tek OCT, frozen and prepared for immunohistochemistry or plated onto laminin- and poly-ornithine-coated plates for 6 days to allow cell migration. Migrating cells were analyzed for the expression of nestin, beta-tubulin III and map2, typical neuronal proteins. Results: DNP-treated EBs exhibited beta-tubulin III and nestin expression levels (18% and 10% positive cells, respectively) similar to the levels obtained using RA (20% and 12% positive cells, respectively). Moreover, DNP-treated EBs exhibited migrating cells expressing both nestin and map2. Further, the migration cell area of DNP-treated EBs was larger than the area of control-treated EBs. Immunoblotting and qPCR analyses will be used to further investigate the expression levels of neuronal-specific proteins in DNP-treated EBs. Conclusion: Results suggest that DNP may be a useful novel drug to induce neural differentiation of mouse embryonic stem cells, providing comparable yields of differentiated cells and greater reproducibility than RA-induced differentiation.