Micropatterning Facilitates the Long‐Term Growth and Analysis of iPSC‐Derived Individual Human Neurons and Neuronal Networks

Abstract

The discovery of induced pluripotent stem cells (iPSC) and their application to patient-specific disease models offers new opportunities for studying the pathophysiology of neurological disorders. However, current methods for culturing iPSC-derived neuronal cells result in clustering of neurons, which precludes the analysis of individual neurons and defined neuronal networks. To address this challenge, we developed cultures of human neurons on micropatterned surfaces that promote neuronal survival over extended periods of time. This approach facilitates studies of neuronal development, cellular trafficking, and related mechanisms that require assessment of individual neurons and specific network connections. Importantly, micropatterns support the long-term stability of cultured neurons, which enables time-dependent analysis of cellular processes in living neurons. The approach described in this paper will allow mechanistic studies of human neurons, both in terms of normal neuronal development and function, as well as time-dependent pathological processes, and will provide a platform for testing of new therapeutics in neuropsychiatric disorders.

Functional Maturation of Human Stem Cell-Derived Neurons in Long-Term Cultures

“…Most culturing conditions for pure stem cell-derived neurons have been based on conditions for primary rodent, primate, and human neurons, and these conditions mostly underperform with stem cell-derived neurons. Different strategies to enable long-term cultures (~100 days) of stem cell or iPSC-derived neurons have been used, such as growing the neurons on micropatterned surfaces [33] and astrocyte co-culture [34]. It has also been shown that adding mouse neurons in addition to astrocytes significantly increases the maturity of induced stem cell-derived neurons [9].…”

“…We successfully grew iNGNs in long-term cultures using astrocyte co-cultures and a simple defined protocol that results in functional neuronal networks. The astrocytes could be helping to “micropattern” the single iNGNs in our studies, since we observed no cell clustering, that hinders long-term cultures [33], and in this way act as physical support for the neurons. The astrocytes also exert their support through secreted factors that benefit neuron viability and synaptogenesis [17,18].…”

μNeurocircuitry: Establishing in vitro models of neurocircuits with human neurons

“…These microfluidic devices have enabled studies of axonal injury 15–17 , axon pathfinding 18 and cellular migration 19 . They have also been utilized to investigate synaptogenesis 20 and circuit connectivity 21–24 . A compartmentalized model of synaptic competition has been devised allowing the study of differences in individual neurons by chemically treating one chamber and performing synapse counting in another 22 .…”

“…have demonstrated the use of optogenetics and calcium imaging as tools to evaluate neuronal communication in a compartmentalized device 21 . These approaches have provided a proof of concept for microfluidics as a strategy to compartmentalize culture in brain circuit studies, but thus far they have only used primary neuronal cultures or are limited to human induced neurons of a single subtype 24 . Furthermore, in most cases, they are not permissive to high-definition functional studies (e.g.…”

Rejuvenation of Cardiac Tissue Developed from Reprogrammed Aged Somatic Cells

“…In addition, it should be noted that all of the observations made in the experiments only provide indirect evidence of whether the 18 M iPSCs were rejuvenated, as aging and senescence can be induced under certain culturing conditions. [34][35][36][37] To determine whether reprogramming of aged somatic cells could lead to rejuvenation, we sought to aggregate 18 M iPSCs and 2 M iPSCs with 8-cell-stage ICR mouse embryos to derive chimeric newborns. Aggregation was performed using three iPSC clones in each group.…”