Introduction: The use of in-vitro neural differentiation (IVND) of human embryonic stem cells (hESCs) is a promising prospective for disease modeling and regenerative medicine. However, it is not yet known when during embryonic development, neurons begin to express functional properties.
Aim: To elucidate the precise developmental timing of intrinsic functional properties of neurons.
Methods
: hESCs were subjected to IVND using the dual SMAD inhibition protocol for the generation of cortical neurons. At 7,14, 21 days following plating onto laminin-coated coverslips , developing neurons were examined morphologically using Cell^A software, in parallel to electrophysiological recordings of Na
+/K
+ currents, spontaneous synaptic activity and evoked action potentials. Data were obtained using Multiclamp amplifier and analyzed using pCLAMP and SigmaPlot software.
Results: 7-days old embryonic neurons were morphologically indistinguishable from neurons at days 14 and 21. However, their excitability, as measured by the ability of cells to fire action potentials (APs), was very low at D7, mild at D14 and high at D21. In accordance, AP threshold and spike duration decreased from D7 to D21. Surprisingly, AP amplitude remained highly uniform. Furthermore, during the 3 week period, a gradual increase in excitability as well as in spontaneous synaptic activity was seen.
Conclusions: Our results demonstrate that although morphologically newborn neurons look mature, their electrical properties develop gradually during IVND. Our results reveal previously unknown aspects regarding the developmental timing of functional neuronal properties and provide a model to track neuronal maturation in-vitro.
