Neonatal mouse cortical but not isogenic human astrocyte feeder layers enhance the functional maturation of induced pluripotent stem cell‐derived neurons in culture

Abstract

Human induced pluripotent stem (iPS) cell-derived neurons and astrocytes are attractive cellular tools for nervous system disease modeling and drug screening. Optimal utilization of these tools requires differentiation protocols that efficiently generate functional cell phenotypes in vitro. As nervous system function is dependent on networked neuronal activity involving both neuronal and astrocytic synaptic functions, we examined astrocyte effects on the functional maturation of neurons from human iPS cell-derived neural stem cells (NSCs). We first demonstrate human iPS cell-derived NSCs can be rapidly differentiated in culture to either neurons or astrocytes with characteristic cellular, molecular and physiological features. Although differentiated neurons were capable of firing multiple action potentials (APs), few cells developed spontaneous electrical activity in culture. We show spontaneous electrical activity was significantly increased by neuronal differentiation of human NSCs on feeder layers of neonatal mouse cortical astrocytes. In contrast, co-culture on feeder layers of isogenic human iPS cell-derived astrocytes had no positive effect on spontaneous neuronal activity. Spontaneous electrical activity was dependent on glutamate receptor-channel function and occurred without changes in I , I , V , and AP properties of iPS cell-derived neurons. These data demonstrate co-culture with neonatal mouse cortical astrocytes but not human isogenic iPS cell-derived astrocytes stimulates glutamatergic synaptic transmission between iPS cell-derived neurons in culture. We present RNA-sequencing data for an immature, fetal-like status of our human iPS cell-derived astrocytes as one possible explanation for their failure to enhance synaptic activity in our co-culture system.

Role of Human-Induced Pluripotent Stem Cell-Derived Spinal Cord Astrocytes in the Functional Maturation of Motor Neurons in a Multielectrode Array System

“…This difference paralleled qPCR profiling showing that the maturation of hiPSC‐MN was less robust when mouse astrocytes were used and pharmacological testing showing only modest responses to drugs acting on neurotransmitter receptors in human/mouse cocultures This appears to be related to species differences rather than regional heterogeneity since rodent astrocytes were derived from the spinal cord. Our findings are in contrast to those of Lischka et al who found that neonatal mouse cortical but not isogenic human astrocyte feeder layers enhanced the maturation of iPSC‐N. It must be noted, however, that those investigators used a cortical patterning protocol to differentiate iPSC‐N, mouse forebrains to derive rodent astrocytes, and a patch‐clamp platform to record neuronal activity.…”

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.…”

In vitro Models for Seizure-Liability Testing Using Induced Pluripotent Stem Cells

“…However, studies using iPSC neurons and rodent astrocytes have highlighted the importance of including astrocytes in culture. Human-neuronal and rodent-astrocytic co-cultures demonstrate enhanced functional maturation, spiking activity, and maintenance of long-term electrical activity cf neurons alone ( Odawara et al, 2014 ; Lischka et al, 2018 ). It has recently been suggested that co-culture of human neurons with rodent astrocytes may generate matured spontaneous electrical activity to a greater extent than using human astrocytes ( Lischka et al, 2018 ).…”