Coculture of Primary Motor Neurons and Schwann Cells as a Model for In Vitro Myelination

Abstract

A culture system that can recapitulate myelination in vitro will not only help us better understand the mechanism of myelination and demyelination, but also find out possible therapeutic interventions for treating demyelinating diseases. Here, we introduce a simple and reproducible myelination culture system using mouse motor neurons (MNs) and Schwann cells (SCs). Dissociated motor neurons are plated on a feeder layer of SCs, which interact with and wrap around the axons of MNs as they differentiate in culture. In our MN-SC coculture system, MNs survived over 3 weeks and extended long axons. Both viability and axon growth of MNs in the coculture were markedly enhanced as compared to those of MN monoculture. Co-labeling of myelin basic proteins (MBPs) and neuronal microtubules revealed that SC formed myelin sheaths by wrapping around the axons of MNs. Furthermore, using the coculture system we found that treatment of an antioxidant substance coenzyme Q10 (Co-Q10) markedly facilitated myelination.

Optogenetic neuronal stimulation promotes axon outgrowth and myelination of motor neurons in a three‐dimensional motor neuron–Schwann cell coculture model on a microfluidic biochip

“…The increased MBP level and enhanced thickness of myelination replicated our previous study involving 2D coculture (Hyung et al, 2015). We tested the effects of optogenetic stimulation at DIV 10 and 20, which we had identified in SCs at the premyelinating and myelinating stages, respectively.…”

“…), as described previously (Hyung et al, 2015;Suh & Hyung, 2018). MNs were harvested from spinal cords (L1-L6 segments) of E13 mouse embryos and cultured in neurobasal/B27 (GibcoA) medium supplemented with 10% horse serum (Gibco), 1% penicillin/ streptomycin (P/S; Sigma), 0.5 mM L-glutamine (Invitrogen), 1 mM β-mercaptoethanol (Sigma), and 50 µg/ml BDNF (Prospec.…”

Myelination key factor krox‐20 is downregulated in Schwann cells and murine sciatic nerves infected by Mycobacterium leprae

“…The relevance of Krox‐20 to SC maturation is unequivocally illustrated in studies on mutations in the EGR2 gene associated with peripheral myelinopathies, including Charcot‐Marie‐Tooth syndrome . Inhibition of Krox‐20 is also observed in non‐hereditary demyelinating neuropathies, such as Guillain‐Barré syndrome . In line, our results demonstrate that ML decreases the frequency of murine SCs expressing Krox‐20 in vivo and in vitro, suggesting that the bacillus may hinder the myelinating phenotype in these cells.…”

Dedifferentiated Schwann cells secrete progranulin that enhances the survival and axon growth of motor neurons

“…We previously showed that the viability of MNs could be markedly enhanced by culturing neurons on top of a confluent feeder layer of SCs (Hyung et al, ). To examine if the increased viability of MNs required a direct cell–cell contact with SCs or was mediated by soluble factors secreted from SCs, we cultured SCs and MNs in a transwell coculture system in which the two types of cells were seeded in different compartments separated by a porous membrane insert (Figure a): MNs were plated and cultured on a coverslip placed in a culture well (bottom compartment), whereas SCs were seeded on a removable transwell membrane insert (upper compartment).…”