Electrical maturation of neurons derived from human embryonic stem cells

Michael Telias,Dalit Ben-Yosef,Menahem Segal

doi:10.12688/f1000research.4943.1

Michael Telias, Dalit Ben-Yosef + Show 1 more

Open Access

https://doi.org/10.12688/f1000research.4943.1

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Abstract

In-vitro neuronal differentiation of human pluripotent stem cells has become a widely used tool in disease modeling and prospective regenerative medicine. Most studies evaluate neurons molecularly and only a handful of them use electrophysiological tools to directly indicate that these are genuine neurons. Therefore, the specific timing of development of intrinsic electrophysiological properties and synaptic capabilities remains poorly understood. Here we describe a systematic analysis of developing neurons derived in-vitro from human embryonic stem cells (hESCs). We show that hESCs differentiated in-vitro into early embryonic neurons, displaying basically mature morphological and electrical features as early as day 37. This early onset of action potential discharges suggests that first stages of neurogenesis in humans are already associated with electrical maturation. Spike frequency, amplitude, duration, threshold and after hyperpolarization were found to be the most predictive parameters for electrical maturity. Furthermore, we were able to detect spontaneous synaptic activity already at these early time-points, demonstrating that neuronal connectivity can develop concomitantly with the gradual process of electrical maturation. These results highlight the functional properties of hESCs in the process of their development into neurons. Moreover, our results provide practical tools for the direct measurement of functional maturity, which can be reproduced and implemented for stem cell research of neurogenesis in general, and neurodevelopmental disorders in particular.

Highlights

In-vitro neural differentiation (IVND) of human pluripotent stem cells, is a promising vehicle for disease modeling and regenerative medicine1–3
Differentiation process and neuronal morphology We have used a slightly modified version of an established protocol for IVND of hESCs5,14, that is based on the dual inhibition of the SMAD pathway (Figure 1A)
Our human pluripotent stem cells (hPSCs) including embryonic stem cells (hESCs) selected for IVND were pluripotent as shown by the expression of OCT4 and the neurons derived were stained positive for MAP2 (>90% of cells), already at day 21 of IVND (Figure 1B)

Summary

Introduction

In-vitro neural differentiation (IVND) of human pluripotent stem cells (hPSCs), is a promising vehicle for disease modeling and regenerative medicine. Upon IVND, confirmation of the neuronal fate is commonly analyzed by the expression of neuron-specific genes, including those of cytoskeletal components (e.g.; TUJ1, MAP2), transcription factors (e.g.; NeuN, NeuroD1) and synaptic proteins (e.g.; synaptotagmin, synaptophysin). Upon IVND, confirmation of the neuronal fate is commonly analyzed by the expression of neuron-specific genes, including those of cytoskeletal components (e.g.; TUJ1, MAP2), transcription factors (e.g.; NeuN, NeuroD1) and synaptic proteins (e.g.; synaptotagmin, synaptophysin)6 The expression of these genes indicates that the cell has acquired the machineries needed to build a neuron, but the ultimate indication that these are genuine neurons involves the analysis of their electrical properties. In the context of human embryonic neurogenesis, analyzing electrical maturation on hPSCs during IVND can shed light over molecular and cellular mechanisms that so far have been studied only using animal models

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