Establishment of a myelinating co-culture system with a motor neuron-like cell line NSC-34 and an adult rat Schwann cell line IFRS1
Co-culture models of neurons and Schwann cells have been utilized for the study of myelination and demyelination in the peripheral nervous system; in most of the previous studies, however, these cells were obtained by primary culture with embryonic or neonatal animals. A spontaneously immortalized Schwann cell line IFRS1 from long-term cultures of adult Fischer rat peripheral nerves has been shown to retain fundamental ability to myelinate neurites in co-cultures with adult rat dorsal root ganglion neurons and nerve growth factor-primed PC12 cells. Our current investigation focuses on the establishment of stable co-culture system with IFRS1 cells and NSC-34 motor neuron-like cells. NSC-34 cells were seeded at a low density (2 × 103/cm2) and maintained for 5–7 days in serum-containing medium supplemented with non-essential amino acids and brain-derived neurotrophic factor (BDNF; 10 ng/mL). Upon observation of neurite outgrowth under a phase-contrast microscope, the NSC-34 cells were exposed to an anti-mitotic agent mitomycin C (1 µg/mL) for 12–16 h, then co-cultured with IFRS1 cells (2 × 104/cm2), and maintained in serum-containing medium supplemented with ascorbic acid (50 µg/mL), BDNF (10 ng/mL), and ciliary neurotrophic factor (10 ng/mL). Double immunofluorescence staining carried out at day 28 of the co-culture showed myelin protein (P0 or PMP22)-immunoreactive IFRS1 cells surrounding the βIII tubulin-immunoreactive neurites. This co-culture system can be a beneficial tool to study the pathogenesis of motor neuron diseases (e.g., amyotrophic lateral sclerosis, Charcot–Marie–Tooth diseases, and immune-mediated demyelinating neuropathies) and novel therapeutic approaches against them.
KeywordsMotor neuron-like cells Immortalized Schwann cells Co-culture Myelination Mitomycin C
This study was supported by a Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (JSPS KAKENHI 16K07048). We would like to thank Dr. Kazuhiko Watabe for providing us NSC-34 cells, Drs. Tatsufumi Murakami, Tomoko Ishibashi and Mari Suzuki for helpful suggestions, and the late Kyoko Ajiki for her enormous contribution to the histochemical analyses.
ST and KS conducted cell culture. ST, HY, NN, and TA conducted immunocytochemical analysis and image presentation. KS, DK, and KU designed the experiments and KS supervised the project. ST and KS drafted the manuscript.
Compliance with ethical standards
Conflict of interest
There is no conflict of interest.
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