Schwann cells are glial cells in the peripheral nervous system that provide trophic support for the growth and maintenance of sensory, motor, and autonomic neurons and ensheath their axons in either a myelinating or an unmyelinating form. Myelinating Schwann cells wrap around large-diameter axons to form multilayered myelin structures essential for the rapid action potential propagation from one node of Ranvier to the next node (saltatory conduction). In contrast, non-myelinating Schwann cells surround several small-diameter axons to form Remak bundles. Following peripheral nerve injury, Schwann cells lose axonal contact and change their phenotype in favor of axonal regeneration and functional restoration. These “de-differentiated” Schwann cells migrate into the site distal to the injury and phagocytose axonal and myelin debris together with macrophages (Wallerian degeneration). Subsequently, they proliferate to constitute the bands of Büngner which act as guideposts for regenerating axons and provide various neurotrophic factors and chemokines that help axonal reinnervation toward target tissues and protect injured neurons from degeneration and cell death. At the final regeneration stage, the “re-differentiated” Schwann cells remyelinate growing large diameter axons or ensheath small diameter axons forming Remak bundles (Sango et al., 2017). Schwann cell abnormalities and/or their crosstalk with neurons lead to demyelinating neuropathy (myelinopathy) development and progression. These deviations are also involved in the manifestations of axons (axonopathy) and neuronal cell bodies (neuronopathy) (Niimi et al., 2019). Cultured Schwann cells and their co-cultures with neuronal cells have been used for myelination and demyelination studies in the peripheral nervous system. In most of the previous studies, however, neurons and Schwann cells were obtained by primary culture (Bolis et al., 2009), which is a time- and energy-consuming process, to get good yields with high cell purity prior to each co-culture experiment. Thus far, some lined Schwann cells have been shown to myelinate neurites in co-cultures with primary cultured neurons (Saavedra et al., 2008). In addition, a myelinating co-culture system with human induced pluripotent stem cell-derived neurons and neonatal rat Schwann cells has been developed (Clark et al., 2017). However, there have been no reports regarding myelination under co-cultures with pure neuronal and Schwann cell lines. The high proliferative activity and phenotypic differences from primary cultured cells may disturb stable neuron-Schwann cell interactions. Spontaneously immortalized Fischer rat Schwann cells 1 (IFRS1) have been established from long-term adult Fischer 344 rat peripheral nerves culture (Sango et al., 2011). IFRS1 cells retain characteristic Schwann cells features, such as spindle-shaped morphology with intense immunoreactivity for Schwann cell markers, such as S100 protein and p75 neurotrophin receptor, mRNA expression of neurotrophic factors (such as nerve growth factor [NGF], glial cell line-derived neurotrophic factor, and ciliary neurotrophic factor) and transcription factors (e.g., Egr2, Sox10, and Oct6). Moreover, IFRS1 cells possess the fundamental capability to myelinate neurites in co-culture with primary cultured adult rat dorsal root ganglion (DRG) neurons. As glial growth stimulant application, such as forskolin and neuregulin-1β, is required for IFRS1 cell passage, the IFRS1 cells excess proliferation can be prevented by the deprivation of these molecules. This feature makes it possible to maintain their co-cultures with not only primary cultured DRG neurons (Sango et al., 2011), but also lined neurons, such as NGF-primed PC12 cells (Sango et al., 2012), rat neural stem cell-derived neurons, mouse embryonic stem (ES) cell-derived motor neurons (Ishii et al., 2017), and NSC-34 motor neuron-like cells (Takaku et al., 2018). Here, the PC12-IFRS1 and NSC-34-IFRS1 co-culture system protocols and features established in our laboratory are briefly described. At the initial trial, undifferentiated PC12 cells (3 × 103/cm2) were directly added to IFRS1 cells (3 × 104/cm2), and the co-cultured cells were maintained in serum-free medium (Dulbecco’s modified Eagle medium [DMEM]/B27 supplement) supplemented with NGF (10 ng/mL); however, this protocol resulted in the excess PC12 cells proliferation. B27 supplement contains numerous nutrients and antioxidants, which might have enhanced the PC12 cells’ proliferative activity even under serum-free culture conditions. PC12 cells were then seeded at a low density (3 × 102/cm2) and maintained in serum-free medium with minimum nutrients (DMEM/N2 supplement; insulin, transferrin, putrescine, selenium. and progesterone) and a high-dose NGF (50 ng/mL). The reduced cell density and the incubation with the minimum nutrients may contribute to the PC12 overgrowth suppression, whereas the NGF-enriched conditions may accelerate their differentiation into neuron-like cells. On day 7 of culture, under these conditions, differentiated PC12 cells were co-cultured with IFRS1 cells (3 × 104/cm2); longer incubation induced PC12 cell death (Sango et al., personal observation), suggesting that the conditions are suitable for differentiation, but not long-term PC12 cell survival. The co-cultured cells were then maintained in DMEM/B27 in the presence of NGF (10 ng/mL), ascorbic acid (50 μg/mL), and soluble neuregulin-1 type III isoform (aka sensory and motor neuron-derived factor, 25 ng/mL). NGF may contribute to neurite elongation and sustenance that emerge from PC12 cells, whereas ascorbic acid is suggested to enhance synthesis of extracellular matrix and basal lamina assembly in Schwann cells. Sensory and motor neuron-derived factor is recognized as a potent myelination-inducible factor and was found to be critical for long-term IFRS1 cell survival in our study (Sango et al., 2012). After 4 weeks of co-culture under this condition, myelin formation was illustrated by immunofluorescence (Figure 1A) and further confirmed by Sudan Black myelin staining and electron microscopy (Sango et al., 2012). The PC12-IFRS1 system is the first co-culture model composed of neuronal and Schwann cell lines that can be prepared at the researchers’ convenience without the need for primary cultures. Despite these advantages, careful attention should be given to prevent co-cultured cell overgrowth and/or death. This system has been utilized for exploring demyelinating neuropathy pathogenesis induced by amiodarone, an anti-arhythmic agent (Niimi et al., 2019), and Schwann cell myelinating activity imaging and demyelinated cell identification using Raman spectroscopy (Pezzotti, 2021). In the former, amiodarone predominantly affected IFRS1 cells through oxidative stress induction and autophagy-lysosome pathway inhibition. Co-cultured cell treatment with amiodarone induced IFRS1 cell detachment from neurite networks in a time- and concentration-dependent manner. The findings that IFRS1 cells are more vulnerable to amiodarone than NGF-primed PC12 cells may account for Schwann cell injury-dominant pathology in amiodarone induced neuropathy. In the latter, real-time Raman analyses were utilized to monitor the myelinating activity of living IFRS1 cells in co-culture with PC12 cells. The Raman imaging at selected wavenumbers may enable the electrochemical processes visualization of myelination and demyelination.Figure 1: Co-culture models with lined neurons and IFRS1 Schwann cells established in the author’s laboratory.(A) A representative double immunofluorescence image of co-culture with PC12 and immortalized Fischer rat IFRS1 Schwann cells at 28 days. Double immunofluorescence was used to illustrate myelin formation using antibodies to myelin protein zero (red) and βIII tubulin (green). Reproduced from Sango et al., 2012 with permission from Springer Nature. (B) Schematic representation of the co-culture models suitable for high throughput screening of the molecules and signaling pathways associated with myelination and demyelination/axonal degeneration (details are described in the text). GLP-1R: Glucagon-like peptide-1 receptor; IFRS1: immortalized Fischer rat Schwann cells 1; NGF: nerve growth factor; SMDF: sensory and motor neuron-derived factor.As it remains controversial if PC12 cells derived from rat pheochromocytoma can be categorized into neurons, the study’s next investigation focused on co-culture system establishment with IFRS1 cells and lined cells that display distinct neuronal phenotypes. NSC-34 cells produced by the fusion of embryonic mouse spinal cord cells with mouse neuroblastoma cells N18TG2 retain some characteristic motor neuron features, such as immunocytochemical choline acetyltransferase expression, synthesis and release of acetylcholine, and acetylcholine receptor cluster formation on co-cultured myotubes. The PC12-IFRS1 co-culture protocol to NSC-34-IFRS1 co-culture was initially applied, but the low cell density and complete serum withdrawal resulted in massive NSC-34 cell death. NSC-34 cells at a higher density (3 × 103/cm2) were then maintained for 7 days in the medium containing 1% fetal bovine serum, non-essential amino acids, and brain-derived neurotrophic factor (10 ng/mL). This condition was effective for NSC-34 cell survival and neurite elongation; however, when they were directly co-cultured with IFRS1 cells, excess NSC-34 cell proliferation occurred disturbing stable IFRS1 cell interactions. Therefore, developing an efficient method to suppress proliferative NSC-34 cell activity was critical for co-culture maintenance. In a study by Acquarone et al. (2015), mouse ES cell pre-treatment with 1 μg/mL mitomycin C (MMC) for 12 hours promoted cell differentiation into the dopaminergic neurons in the absence of apoptotic cell death. In addition, intra-striatal injection of the MMC-treated ES cells restored motor function without teratoma formation in a Parkinson’s disease murine model. In a similar manner to that study, we observed that MMC pre-treatment (1 μg/mL for 12–16 hours) was observed to have effectively prevented the excess proliferation or death of NSC-34 cells after they were co-cultured with IFRS1 cells. Finally, the co-cultured cells were maintained in the medium containing 5% fetal bovine serum, ascorbic acid (50 μg/mL), brain-derived neurotrophic factor (10 ng/mL), and ciliary neurotrophic factor (10 ng/mL). This serum-rich condition with the neuroprotective molecules alleviated MMC toxicity and allowed most of NSC-34 cells to survive for > 3 weeks. After 4 weeks of co-culture, myelin formation was illustrated by immunofluorescence (Takaku et al., 2018). By utilizing this protocol with slight modifications, a stable co-culture system with IFRS1 cells and ND7/23 cells (a hybridoma of neonatal rat DRG neurons and mouse neuroblastoma cells N18TG2) was recently established (Takaku et al., in preparation). ND7/23 cells displayed some characteristic sensory neuron features, such as immunocytochemical expression of substance P and high-affinity NGF receptor TrkA, and formaldehyde-induced increases in intracellular Ca2+. In summary, stable co-culture systems with IFRS1 Schwann cells and lined neurons have been established, such as NGF-primed PC12 cells, NSC-34 motor neuron-like cells, and ND7/23 sensory neuron-like cells. These systems are suitable for high throughput screening of the molecules and pathways involved in axonal regeneration and remyelination after injury and development and progression of peripheral neuropathies (Figure 1B). By using the DRG neuron-IFRS1 co-culture system, the exendin-4 (Ex-4) neurotrophic and neuroprotective activities were recently demonstrated, a glucagon-like peptide-1 receptor agonist utilized as an anti-diabetic agent (Takaku et al., 2021); Ex-4 (100 nM) accelerated DRG neuron neurite outgrowth, IFRS1 cell survival and migration, and myelin formation in their co-cultures with upregulation of the expression of phosphorylated serine/threonine-specific protein kinase AKT. In addition, Ex-4’s beneficial effects on DRG neurons and IFRS1 cells were attenuated by LY294002, a phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor. These findings suggest that Ex-4 binds to glucagon-like peptide-1 receptor in both DRG neurons and Schwann cells to accelerate the myelination process through the PI3K/AKT signaling pathway activation. To elucidate more precise action mechanisms of Ex-4 and pursue its potential repositioning toward peripheral nerve lesions, this team is about to survey the PI3K/AKT pathway downstream target molecules (e.g., cyclic AMP response element-binding protein, mammalian target of rapamycin, and ras homolog family member A) by using ND7/23-IFRS1 co-culture model and multi-omics approaches. Moreover, it is expected that NSC-34-IFRS1 and ND7/23-IFRS1 co-culture models will be beneficial tools to study the degeneration and regeneration of the motor and sensory nervous systems, respectively. This team’s ongoing study with these models is aimed at elucidating the pathogenesis of drug-induced demyelinating neuropathies (amiodarone, dichloroacetate, immune checkpoint inhibitors, etc.) (Niimi et al., 2019). Furthermore, future projects in collaboration with neuroimmunologists may contribute to the pathogenesis-based development of remedies toward immune mediated demyelinating neuropathies, such as Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, and immunoglobulin M anti-myelin associated glycoprotein neuropathy. C-Editors: Zhao M, Liu WJ, Li CH; T-Editor: Jia Y
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Apr 25, 2024
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