Myelination in coculture of established neuronal and Schwann cell lines

Abstract

Establishing stable coculture systems with neuronal and Schwann cell lines has been considered difficult, presumably because of their high proliferative activity and phenotypic differences from primary cultured cells. The present study is aimed at developing methods for myelin formation under coculture of the neural crest-derived pheochromocytoma cell line PC12 and the immortalized adult rat Schwann cell line IFRS1. Prior to coculture, PC12 cells were seeded at low density (3 × 10(2)/cm(2)) and maintained in serum-free medium with N2 supplement, ascorbic acid (50 μg/ml), and nerve growth factor (NGF) (50 ng/ml) for a week. Exposure to such a NGF-rich environment with minimum nutrients accelerated differentiation and neurite extension, but not proliferation, of PC12 cells. When IFRS1 cells were added to NGF-primed PC12 cells, the cell density ratio of PC12 cells to IFRS1 cells was adjusted from 1:50 to 1:100. The cocultured cells were then maintained in serum-free medium with B27 supplement, ascorbic acid (50 μg/ml), NGF (10 ng/ml), and recombinant soluble neuregulin-1 type III (25 ng/ml). Myelin formation was illustrated by light and electron microscopy performed at day 28 of coculture. The stable PC12-IFRS1 coculture system is free of technical and ethical problems arising from the primary culture and can be a valuable tool to study peripheral nerve degeneration and regeneration.

Involvement of oxidative stress and impaired lysosomal degradation in amiodarone‐induced schwannopathy

“…The average number of attached Schwann cells is 1.4 ± 0.7 in [AMD 10 μ m ] and 3.5 ± 2.9 in [Control], respectively ( n ≥ 15 neurites from 3 coculture samples); the former is significantly lower than the latter (unpaired Student's t ‐test, t 44 = 3.957, P = 0.0003). Because we had previously verified the formation of myelin in these coculture systems (Sango et al ., , ), these findings implicate AMD as a trigger of demyelination. To further confirm that these morphological changes are the result of demyelination, we conducted Sudan black B staining and electron microscopy on the cocultures; however, the AMD‐treated samples were much more fragile than the untreated ones and totally detached from the Aclar coverslips during dehydration with graded ethanol (Niimi et al ., personal observation).…”

“…In contrast, AMD at higher doses (≥ 20 μ m ) and/or for longer exposure time (48–72 h) was required to induce massive IFRS1 cell detachment from the neurites of PC12 cells. This may be because of the higher density of IFRS1 cells at the start of the coculture and the maintenance of the coculture with SMDF, which enhanced the viability of IFRS1 cells (Sango et al ., ). Moreover, we observed similar morphological changes in the primary Schwann cells of DRG under exposure to AMD (Fig.…”

“…Cocultures of IFRS1 cells and PC12 cells were performed as described previously with slight modifications (Sango et al, 2012). Briefly, PC12 cells were detached from type I collagen-coated 100mm dishes by gentle pipetting and reseeded on type I collagencoated Aclar coverslips at an approximate density of 5 9 10 2 /cm 2 .…”

“…IFRS1 cells exhibit distinct Schwann cell phenotypes, such as spindle-shaped morphology under phase-contrast microscopy, expression of Schwann cell markers (e.g. S100 and p75 low affinity neurotrophin receptor), synthesis and secretion of neurotrophic factors and cytokines (Sango et al, 2011;Takaku et al, 2013), and fundamental ability to myelinate neurites in cocultures with DRG neurons (Sango et al, 2011) and nerve growth factor (NGF)-primed PC12 cells (Sango et al, 2012). Consequently, IFRS1 cells and their cocultures with neuronal cells can be beneficial tools to help characterize AMD neurotoxicity.…”

Immortalized Schwann cells IFRS1 as a new strategic tool for the study of myelination and demyelination

“…Namely, the absence of these molecules suppressed excess proliferation of IFRS1 cells and made it possible to maintain their coculture with adult rat DRG neurons (Sango et al, 2011a) and NGF-primed PC12 cells (Sango et al, 2012a).…”