Reproducible and efficient generation of functionally active neurons from human hiPSCs for preclinical disease modeling

Abstract

The use of human induced pluripotent stem cell (hiPSC)-derived neuronal cultures to study the mechanisms of neurological disorders is often limited by low efficiency and high variability in differentiation of functional neurons. Here we compare the functional properties of neurons in cultures prepared with two hiPSC differentiation protocols, both plated on astroglial feeder layers. Using a protocol with an expandable intermediate stage, only a small percentage of cells with neuronal morphology were excitable by 21–23 days in culture. In contrast, a direct differentiation strategy of the same hiPSC line produced cultures in which the majority of neurons fired action potentials as early as 4–5 days. By 35–38 days over 80% of the neurons fired repetitively and many fired spontaneously. Spontaneous post-synaptic currents were observed in ~40% of the neurons at 4–5 days and in ~80% by 21–23 days. The majority (75%) received both glutamatergic and GABAergic spontaneous postsynaptic currents. The rate and degree of maturation of excitability and synaptic activity was similar between multiple independent platings from a single hiPSC line, and between two different control hiPSC lines. Cultures of rapidly functional neurons will facilitate identification of cellular mechanisms underlying genetically defined neurological disorders and development of novel therapeutics.

A Temperature-Controlled Patch Clamp Platform Demonstrated on Jurkat T Lymphocytes and Human Induced Pluripotent Stem Cell-Derived Neurons

“…This feature of higher maximal firing frequency was reproducible and goes along with a reduced AP height, summarized in Figure 5 d,e. The averaged maximal firing frequency increased from 20 to 36 Hz while the corresponding height decreased from 80.8 to 54.2 mV—in general appropriate values for AP heights [ 20 , 35 , 44 , 45 ]. The higher frequency can be explained by faster gating ion channels again allowing for a more rapid sequence of depolarization and repolarization of the cell membrane.…”

Stem cell models of human synapse development and degeneration

“…Addition of astrocytes to neuronal cultures promotes synapse maturation and spontaneous action potential formation. This can be accomplished through 1) cultures that generate both neurons and astrocytes through a shared progenitor, 2) hIPSC-derived neuron-astrocyte cocultures, or 3) application of astrocyte-conditioned media to hIPSC-derived neurons (Tang et al , 2013; Odawara et al , 2016; Gunhanlar et al , 2018; Xie et al , 2018). Defined cocultures can also be used to address how specific cell types contribute to disease progression.…”

Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks

“…Various studies have aimed to test neuron-astrocyte interactions using in vitro co-culture systems. These have included testing for interactions during development, such as the astrocytic effect on differentiation of precursor cells and stem cells (Johnson et al, 2007 ; Tang et al, 2013 ; Ehret et al, 2015 ; Lischka et al, 2017 ; Xie et al, 2017 ; Schutte et al, 2018 ), synchronization of neuronal network activity (Kuijlaars et al, 2016 ), and number of formed synapses (Pyka et al, 2011 ; Jones et al, 2012 ; Shi et al, 2013 ), and also the effect during disease states, such as in ALS (Kunze et al, 2013 ), thymine deficiency (Park et al, 2001 ), oxidative stress (Kidambi et al, 2008 ), and general neurotoxicity (Anderl et al, 2009 ). While the relevance of co-cultures of neurons and astrocytes has become more evident as more and more functions of astrocytes are discovered, their cumulative effect on the survival of low-density networks is seldom addressed and remains unclear.…”