Generation of Active Neurons from Mouse Embryonic Stem Cells Using Retinoic Acid and Purmorphamine

Multiple differentiation protocols have emerged in recent years, producing neurons with diverse morphologies, gene and protein expression profiles, and functionality. Many of these differentiation techniques require months of culture and the use of expensive growth factors. Most importantly, the derived neurons usually do not exhibit any electrical activity. This limits the value of the protocol as a tool for engineering and investigating neural networks. Here, we describe an efficacious method for differentiating mouse embryonic stem cells into functional neurons. CGR8 cells were neurally induced via the simultaneous application of retinoic acid and purmorphamine. The derived cells expressed neuronal (TUJ1 and NeuN) and synaptic (GAD2, PSD-95, Synaptophysin, and VGLUT1) markers. During whole-cell recordings, neurons exhibited inward and outward currents, likely caused by fast-inactivating voltage-gated potassium channels. Upon current injection, miniature action potentials were also recorded. The efficient generation of diverse subtypes of functional neurons can be a useful tool in fundamental investigations of neural network activity and translational studies.