Stem cell derived neurons : physiology of early differentiation and development

The developing nervous system of vertebrates is largely inaccessible; especially early neuronal differentiation and development are difficult to study. Early events in neurogenesis can be investigated more easily by using murine embryonic stem (mES) cells. Under suitable culture conditions mES cells pass through all phases of neural differentiation through an intermediate stage of embryoid bodies. As wild type or genetically modified ES cells can be grown in unlimited quantities, their differentiation into neurons represents an attractive model for studying neurodevelopmental diseases. Functional neuronal maturation can now be followed from immature to mature neurons forming excitatory and inhibitory synaptically connected networks.
In my thesis work I used such a culture system to compare the functional development of mES cell derived neurons from different genetic backgrounds. I spent a lot of time establishing experimental conditions to study these cells using electrophysiological techniques. I quantified several key parameters such as resting membrane potential, voltage-gated channel activity, appearance of action potentials, to describe the neuronal differentiation of the cells. In the first days after seeding the precursors possess an immature physiology, with high input resistances, few voltage-sensitive conductance and immature spiking patterns. During subsequent development an increasing amount of voltage gated sodium- and potassium currents appeared, leading to more and more mature spiking patterns. Mature neurons form synaptically connected networks containing excitatory and inhibitory neurons. We investigated the development of spontaneous excitatory- and inhibitory postsynaptic currents (sEPSCs and sIPSCs) in these cultures. We then built upon this knowledge of normal development to study mES cell-derived neurons defective in MeCP2 signaling. This genetic defect causes an important neurodevelopmental defect in mice and in humans. We were able to show that key features of the developmental defect – a disturbed maturation of inhibitory synapses – could be reproduced in our system. For further characterization of inhibitory interneurons we derived stem cells from a GAD67-GFP cell line under two pharmacological conditions as KCl and TTX which change the activity level up or down. This will aid to understand more about the developmental processes of neurons and will help finding functional deficits in neurodevelopmental diseases.