Establishment of a myelinating co-culture system with a motor neuron-like cell line NSC-34 and an adult rat Schwann cell line IFRS1

Abstract

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 × 10/cm) 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 × 10/cm), 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.

Models and methods to study Schwann cells

“…(Chen et al, 2019c ; Negro et al, 2016 , 2018 ) (Figure 4b ). For example, myelination has been extensively studied in normal and pathological conditions using co‐cultures of DRG neurons and SCs (Chan et al, 2004 ; Trimarco et al, 2014 ; Zhang et al, 2009 ), or by co‐culturing immortalized SC cell lines and adult rat DRG or MN‐like cells (Eldridge et al, 1987 ; Sango et al, 2011 ; Takaku et al, 2018 ). In the first approach, SCs are plated to seeded DRG neurons.…”

Glucagon-Like Peptide-1 Receptor Agonists as Potential Myelination-Inducible and Anti-Demyelinating Remedies

“…Co-culture systems of neurons and Schwann cells have enabled us to generate myelin structures nearly equivalent to those in living bodies, thereby being recognized as useful tools to investigate neuron-Schwann cell interactions ( Ogata et al, 2004 ). Immortalized adult Fischer rat Schwann cells 1 (IFRS1) established in our laboratory display distinct Schwann cell phenotypes, including fundamental ability to myelinate neurites in co-culture with adult rat dorsal root ganglion (DRG) neurons ( Sango et al, 2011 ), nerve growth factor-primed PC12 cells ( Sango et al, 2012 ), and NSC-34 motor neuron-like cells ( Takaku et al, 2018 ). As compared with the previous co-culture models using embryonic and/or neonatal animals, our DRG neuron-IFRS1 co-culture system has the following advantages: 1) Both neurons and Schwann cells are derived from adult rats and retain the biological properties of the mature peripheral nervous system.…”

Lost in Translation? Measuring Diabetic Neuropathy in Humans and Animals

“…It is also worth noting that most culture media used to sustain neurons already contains glucose concentrations of 20 mM or more, this being in marked excess of those considered hyperglycemic in vivo, making experimental design and interpretation difficult. Sophisticated cell culture systems employ cells derived from adult normal and diabetic animals that are excised and maintained under in vitro conditions that reflect the source glycemic and insulin environment [90] and co-culture Schwann cells and neurons to promote myelination [91,92].…”