Myelinating cocultures of rodent stem cell line‐derived neurons and immortalized Schwann cells

Abstract

Myelination is one of the most remarkable biological events in the neuron-glia interactions for the development of the mammalian nervous system. To elucidate molecular mechanisms of cell-to-cell interactions in myelin synthesis in vitro, establishment of the myelinating system in cocultures of continuous neuronal and glial cell lines are desirable. In the present study, we performed co-culture experiments using rat neural stem cell-derived neurons or mouse embryonic stem (ES) cell-derived motoneurons with immortalized rat IFRS1 Schwann cells to establish myelinating cultures between these cell lines. Differentiated neurons derived from an adult rat neural stem cell line 1464R or motoneurons derived from a mouse ES cell line NCH4.3, were mixed with IFRS1 Schwann cells, plated, and maintained in serum-free F12 medium with B27 supplement, ascorbic acid, and glial cell line-derived neurotrophic factor. Myelin formation was demonstrated by electron microscopy at 4 weeks in cocultures of 1464R-derived neurons or NCH4.3-derived motoneurons with IFRS1 Schwann cells. These in vitro coculture systems utilizing the rodent stable stem and Schwann cell lines can be useful in studies of peripheral nerve development and regeneration.

Praja1 RING‐finger E3 ubiquitin ligase suppresses neuronal cytoplasmic TDP‐43 aggregate formation

“…In the present study, we used an adult rat neural stem cell line 1464R that we established to examine aggregate formation of adenovirally‐induced TDP‐43 as described previously 8,9,23 . The 1464R cells differentiate predominantly (> 80%) into TuJ1‐positive neurons and, to a lesser extent (< 20%), into glial fibrillary acidic protein (GFAP)‐positive astrocytes and O4‐positive oligodendrocytes in the presence of ATRA 8,9,23 .…”

“…All experiments were performed in accordance with Japanese National Guidelines and Regulations, and were approved by the Biosafety Committee and the Animal Care and Use Committee of the Kyorin University Faculty of Health Sciences. The 1464R adult rat neural stem cell line was established as previously described 8,9,23 (#T0746; Applied Biological Materials, Richmond, Canada) and cells were cultured in growth medium composed of Neurobasal medium (#21103-049; Thermo Fisher Scientific, Waltham, CA, USA) containing 2 mM L-glutamine (#25030-081; Thermo Fisher), 2% B27 supplement (#17504-044; Thermo Fisher), 10 ng/mL fibroblast growth factor 2 (FGF2) (#F0291; Sigma, St. Louis, MO, USA), 10 ng/mL epidermal growth factor (EGF) (#E9644; Sigma), 50 units/ mL penicillin and 50 μg/mL streptomycin (#15070-063; Thermo Fisher) on 10-cm dishes coated with poly(2hydroxyethylmethacrylate) (PHEMA) (#P3932; Sigma) to prevent cell attachment. Cells were maintained in 5% CO 2 at 37 C. The 1464R cells formed typical neurospheres, and were mechanically dissociated, serially passaged in the same medium twice a week, and cultured further for more than 50 passages with no obvious morphological alterations.…”

“…In the present study, we used an adult rat neural stem cell line 1464R that we established to examine aggregate formation of adenovirally-induced TDP-43 as described previously. 8,9,23 The 1464R cells differentiate predominantly (> 80%) into TuJ1-positive neurons and, to a lesser extent (< 20%), into glial fibrillary acidic protein (GFAP)positive astrocytes and O4-positive oligodendrocytes in the presence of ATRA. 8,9,23 These differentiated 1464R neurons and glial cells formed cytoplasmic aggregates of TDP-43 following co-infection of adenoviruses expressing DsRed-tagged WT and CTF TDP-43 in the presence of proteasome inhibitors.…”

“…8,9,23 The 1464R cells differentiate predominantly (> 80%) into TuJ1-positive neurons and, to a lesser extent (< 20%), into glial fibrillary acidic protein (GFAP)positive astrocytes and O4-positive oligodendrocytes in the presence of ATRA. 8,9,23 These differentiated 1464R neurons and glial cells formed cytoplasmic aggregates of TDP-43 following co-infection of adenoviruses expressing DsRed-tagged WT and CTF TDP-43 in the presence of proteasome inhibitors. 8,9 These cytoplasmic TDP-43 aggregates were strongly immunoreactive for phosphorylated TDP-43, p62, and ubiquitin, were ultrastructurally composed of non-membrane bound, electron-dense fine granular materials, and were detected in sarkosyl-insoluble or RIPA-insoluble fraction by Western blot analysis, which recapitulates pathological features of TDP-43 aggregates observed in human ALS and FTLD.…”

Generation of High-Yield, Functional Oligodendrocytes from a c-myc Immortalized Neural Cell Line, Endowed with Staminal Properties

“…Although it is possible to generate NSCs from embryonic and even an adult brain, some drawbacks such as reproducibility, time and expensive experimental procedures, hamper their use as a biomedical tool. Therefore, established cell lines with staminal properties are a useful tool [29][30][31]. A1 cells…”