Most studies focusing on neural development and the mechanism of neurodevelopmental disorders are based on animal (rodent or avian) models or post mortem human samples. Thus, the observations derived from these studies cannot be directly translated into mechanistic insights of human neural development and regeneration. Human pluripotent stem cells, which can potentially be differentiated into any cell types of the body, offer a new perspective to overcome this limitation. In the present study, we differentiated induced pluripotent stem cell lines toward the neural lineage. Using a multi‐step, directed differentiation protocol, specific human neural progenitor cells (NPCs) were generated which are committed to produce PROX‐1‐positive granule cells of dentate gyrus of the hippocampus. These NPCs were characterized in terms of morphology, expression of specific cellular markers, growth capacity, and capability of producing mature neurons. Next we investigated the calcium signals in NPCs and PROX1‐positive hippocampal neurons, expressing GCaMP6fast calcium indicator protein or loaded with Fluo4 calcium sensitive dye, in parallel experiments. We demonstrated the functional presence of several receptors, and also the lack of functioning sodium channels in NPCs, and spontaneous Ca2+ gradients in neurons by both methods. We found that spontaneous calcium gradients and specific, ligand induced calcium signals were not significantly altered in the GCaMP6f expressing cells, suggesting the applicability of GCaMP6f for the investigation of fast calcium signals in neural cultures. We conclude that the characteristics of both the spontaneous and ligand‐induced Ca2+‐signals, as well as their pharmacological modulation can be successfully examined in these human model cells by fluorescence imaging.
Á. Apáti, Tünde Berecz, Eszter Szabó
The FASEB Journal