Role of human induced pluripotent stem cell-derived spinal cord astrocytes in the functional maturation of motor neurons in a multielectrode array system

Abstract

The ability to generate human induced pluripotent stem cell (hiPSC)-derived neural cells displaying region-specific phenotypes is of particular interest for modeling central nervous system (CNS) biology in vitro. We describe a unique method by which spinal cord hiPSC-derived astrocytes (hiPSC-A) are cultured with spinal cord hiPSC-derived motor neurons (hiPSC-MN) in a multielectrode array (MEA) system to record electrophysiological activity over time. We show that hiPSC-A enhance hiPSC-MN electrophysiological maturation in a time-dependent fashion.The sequence of plating, density, and age in which hiPSC-As are co-cultured with MN, but not their respective hiPSC line origin, are factors that influence neuronal electrophysiology. When compared to co-culture with mouse primary spinal cord astrocytes, we observe an earlier and more robust electrophysiological maturation in the fully human cultures, suggesting that the human origin is relevant to the recapitulation of astrocyte/motor neuron cross-talk. Finally, we test pharmacological compounds on our MEA platform and observe changes in electrophysiological activity which confirm hiPSC-MN maturation. These findings are supported by immunocytochemistry and real time PCR studies in parallel cultures demonstrating human astrocyte mediated changes in the structural maturation and protein expression profiles of the neurons. Interestingly, this relationship is reciprocal and co-culture with neurons influences astrocyte maturation as well. Taken together these data indicate that in a human in vitro spinal cord culture system, astrocytes alter hiPSC-MN maturation in a time-dependent and species specific manner and suggest a closer approximation of in vivo conditions. Main Points1. We developed a method for the co-culture of human iPSC-A/MN for multielectrode array recordings.2. The morphological, molecular, pharmacological, and electrophysiological characterization of the co-cultures suggests bidirectional maturation.

An optimized workflow to generate and characterize iPSC-derived motor neuron (MN) spheroids

“…The cell bodies of the MNs often form clusters with their axons entangled (Fig 1A ), making them inaccessible for some analyses. For instance, monitoring cell morphology, electrical activity and protein expression through immunocytochemistry becomes a challenging task (25,26). Moreover, the clustering cells are prone to detachment from the surface, preventing their long-term culture.…”