Abstract
Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48–55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time.
Highlights
Stem cell biology has great potential for the study and treatment of neurodegenerative diseases [1]
Immunofluorescent examination of induced pluripotent stem cells (iPSCs)-derived neurons Neurons were generated from iPSCs using a standard method that relies on the inhibition of both branches of TGFb signaling in undifferentiated pluripotent cells [17,37]
Similar to human embryonic stem cells (ESCs), human iPSCs derived from somatic cells possess self-renewal and pluripotency properties and are expected to serve as a powerful tool to model diseases for basic and translational research [58,59,60,61,62]
Summary
Stem cell biology has great potential for the study and treatment of neurodegenerative diseases [1]. Fibroblasts can be transdifferentiated directly to neurons [14,18] Neurons generated from these reprogramming protocols clearly express markers reflecting their relative stage of differentiation, such as nestin [19,20], b-III tubulin [12,21], MAP2 [22,23] NeuN [24], synapsin 1 [25] and synaptophysin [24,26], indicating physiological neuronal development. A few studies have investigated the evolution of the electrophysiological properties of murine iPSC-derived neurons during their maturation from progenitors in mice or rats in vivo or in vitro [28,29,30], these works have not been fully replicated using human iPSCderived neurons. Some recent studies have followed the modification of the basal electrophysiological properties of the cells during their differentiation into neurons and their maturation, focusing on particular protocols of differentiation [27] or on the comparison between ESCs and iPSCs [31]
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