Polyethyleneimine facilitates the growth and electrophysiological characterization of motor neurons

Abstract

Abstract Background Induced pluripotent stem cell (iPSC) technology, in combination with electrophysiological characterization via multielectrode array (MEA), has facilitated the utilization of iPSC-derived motor neurons (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. Methods Human iPSCs were differentiated into MNs using an existing and our own novel protocols, and MNs were cultured on dishes coated with different coating conditions, including. POL (20µg/ml Poly-L-ornithine and 20µg/ml Laminin), POLF (20µg/ml Poly-L-ornithine, 20µg/ml Laminin and 10µg/ml Fibronectin), POLFM (20µg/ml Poly-L-ornithine, 20µg/ml Laminin, 10µg/ml Fibronectin and 1:20 Matrigel), POM (20µg/ml Poly-L-ornithine and 1:50 Matrigel), and PEI (0.1% Polyethyleneimine in borate buffer). We comparatively evaluated cell morphology, aggregation status and electrophysiological activities by MEA. Results 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-derived MNs. We showed that two coating conditions, namely, POM and PEI strongly promoted attachment of iPSC-derived sMNs on MEA culture dishes compared to the other three conditions, and both facilitated the maturation of iPSC-derived sMNs 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-sMNs in 7-week cultures, which permitted the detection of enhanced excitability in iPSC-derived sMNs from patients with amyotrophic lateral sclerosis (ALS). Conclusion This study provides a comprehensive comparison of five coating conditions and offers POM and PEI as favorable coatings for in vitro modeling of neurodevelopmental and neurodegenerative disorders, respectively.