Research Article Open Access

Polyethyleneimine Facilitates the Growth and Electrophysiological 2 Characterization of iPSC-Derived Motor Neurons

Abstract

Induced Pluripotent Stem Cell (iPSC) technology, in combination with electrophysiological characterization via Multielectrode Array (MEA), has facilitated the utilization of iPSC-derived Motor Neurons (iPSC-MNs) as highly valuable models for underpinning pathogenic mechanisms and developing novel therapeutic interventions for Motor Neuron Diseases (MNDs). However, the challenge of adherence of MNs to MEA plates and the heterogeneity presented in iPSC-derived cultures raise concerns about the reliability of the findings obtained from these cellular models. We discovered that one factor modulating the electrophysiological activity of iPSC-MNs is the Extracellular Matrix (ECM) used in coating to support the in vitro growth, differentiation and maturation of iPSC-MNs. This study here showed that two coating conditions, namely, Poly-L-ornithine/Matrigel (POM) and Polyethyleneimine (PEI) strongly promoted attachment of iPSC-MNs on MEA culture dishes compared to the other three conditions and both facilitated the maturation of iPSC-MNs as characterized by the detection of extensive electrophysiological activities from the MEA plates. POM coating accelerated the maturation of the iPSC-MNs for up to 5 weeks, which facilitates the modeling of neurodevelopmental disorders. However, the application of PEI resulted in more even distribution of the MNs on the culture dish and reduced variability of electrophysiological signals from the iPSC-MNs in 7-week cultures, which permitted the detection of enhanced excitability in iPSC-MNs from patients with Amyotrophic Lateral Sclerosis (ALS). This study provides a comprehensive comparison of five coating conditions and offers POM and PEI as favourable coatings for in vitro modelling of neurodevelopmental and neurodegenerative disorders, respectively. Significant statement: Improvement of motor neuron differentiation protocol and production of highly pure functional Motor Neurons (MNs) are essential for modelling motor neuron diseases, drug screening and cell replacement therapy. However, the coating matrix can significantly affect the in vitro performance of motor neurons. In this study, we provided a comprehensive comparison of five coating conditions and offers POM and PEI as favourable coating for in vitro modelling neurodevelopment and neurodegenerative disorders. This also permitted identification of increased excitability phenotypes in the sporadic ALS iPSCs-derived spinal MNs using both POM and PEI coating. Therefore, this novel protocol and derived functional MNs may find a wide application in disease modelling, drug discovery and cell therapy. Meimei Yang, Daofeng You, Gang Liu, Yin Lu, Guangming Yang, Timothy O'Brien, David C Henshall, Orla Hardiman, Min Liu, Sanbing Shen

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