A Time Course Analysis of the Electrophysiological Properties of Neurons Differentiated from Human Induced Pluripotent Stem Cells (iPSCs)

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.

Being human: The role of pluripotent stem cells in regenerative medicine and humanizing Alzheimer's disease models

“…PSC-derived AD neurons would require very mature neuronal networks to assay changes in long-term potentiation (LTP) and depression (LTD) as measures of synaptic plasticity. We have recently published a study describing the evolution of electrophysiological properties of iPSC-derived neurons, which could be used as a baseline for studies seeking to improve electrophysiological maturity (Pre et al, 2014). In more broad terms, early onset AD still takes at least 18 years to manifest in humans, which would be an awfully long experiment in a dish.…”

Characterization of a subpopulation of developing cortical interneurons from human iPSCs within serum-free embryoid bodies

“…The identification of 108 this group of cells within SFEBs consisting of iPSCs is an important and novel step in 109 directing cortical development in this system and refining tools for disease modeling. iPSCs cell lines, and culture and differentiation methods have been previously described 114 [35,38,42] deplete MEFs, cells were plated for 1 hour on gelatin-coated culture dishes. 126 127 Remaining cells were transferred to a 96-well V-bottom plate (Oregon Scientific) at a 128 density of 9,000 cells per well in DMEM/F12 containing the dual-SMAD inhibitors 129 SB431542 (10 M) and LDN193189 (250 nM, Stemgent) and recombinant Dickkopf-1 130 (Dkk-1) (R&D Systems) (200 ng/mL) [10,22] .…”

“…The perfusion chamber contained an extracellular 182 solution consisting of (in mM): NaCl (119), KCl (5), CaCl 2 (2), 2 MgCl 2 , Sucrose (40), (EGTA) (0.6), and HEPES (5) titrated to a pH of 7.1 and an osmolarity of 310 mOsm [38] . 188…”

Advances and Current Challenges Associated with the Use of Human Induced Pluripotent Stem Cells in Modeling Neurodegenerative Disease

“…Regardless of the AP properties of individual neurons, the predictive value of iPSC-based central nervous system models is predicated on the ability of the derived cells to recapitulate accurately the functional behaviors of primary neuronal networks through synaptic connectivity. Neurons derived from human iPSCs have been shown to fire AP in response to depolarizing current injections after just 3 weeks of differentiation [Prè et al, 2014]. However, the AP properties at this time point are relatively poor, but in the following weeks they undergo significant improvements characterized by increases in Na + and K + current amplitudes, AP amplitude, full width at half maximum of the AP spike, and AP threshold [Prè et al, 2014].…”

“…Neurons derived from human iPSCs have been shown to fire AP in response to depolarizing current injections after just 3 weeks of differentiation [Prè et al, 2014]. However, the AP properties at this time point are relatively poor, but in the following weeks they undergo significant improvements characterized by increases in Na + and K + current amplitudes, AP amplitude, full width at half maximum of the AP spike, and AP threshold [Prè et al, 2014]. Resting membrane potential in these cells likewise decreased with time, illustrating an improved capacity for cells to regulate ion gradients across their membranes with increased time in culture.…”