A microfabricated multi-compartment device for neuron and Schwann cell differentiation.
Ontology highlight
ABSTRACT:
SUBMITTER: De Vitis E
PROVIDER: S-EPMC8007719 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Myelinating Schwann cell (SC)-dorsal root ganglion (DRG) neuron cocultures are an important technique for understanding cell-cell signalling and interactions during peripheral nervous system (PNS) myelination, injury, and regeneration. Although methods using rat SCs and neurons or mouse DRG explants are commonplace, there are no established protocols for compartmentalised myelinating cocultures with dissociated mouse cells. There consequently is a need for a coculture protocol that allows separate genetic manipulation of mouse SCs or neurons, or use of cells from different transgenic animals to complement in vivo mouse experiments. However, inducing myelination of dissociated mouse SCs in culture is challenging. Here, we describe a new method to coculture dissociated mouse SCs and DRG neurons in microfluidic chambers and induce robust myelination. Cocultures can be axotomised to study injury and used for drug treatments, and cells can be lentivirally transduced for live imaging. We used this model to investigate axon degeneration after traumatic axotomy and find that SCs, irrespective of myelination status, are axo-protective. At later timepoints after injury, live imaging of cocultures shows that SCs break up, ingest and clear axonal debris.
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Project description:Formation of myelin sheaths by oligodendrocytes (OLs) in the central nervous system (CNS) is essential for rapid nerve impulse conduction. Reciprocal signaling between axons and OLs orchestrates myelinogenesis but remains largely elusive. In this study, we present a multi-compartment CNS neuron-glia microfluidic co-culture platform. The platform is capable of conducting parallel localized drug and biomolecule treatments while carrying out multiple co-culture conditions in a single device for studying axon-glia interactions at a higher throughput. The "micro-macro hybrid soft-lithography master fabrication" (MMHSM) technique enables a large number of precisely replicated PDMS devices incorporating both millimeter and micrometer scale structures to be rapidly fabricated without any manual reservoir punching processes. Axons grown from the neuronal somata were physically and fluidically isolated inside the six satellite axon/glia compartments for localized treatments. Astrocytes, when seeded and co-cultured after the establishment of the isolated axons in the satellite axon/glia compartments, were found to physically damage the established axonal layer, as they tend to grow underneath the axons. In contrast, oligodendrocyte progenitor cells (OPCs) could be co-cultured successfully with the isolated axons and differentiated into mature myelin basic protein-expressing OLs with processes aligning to neighboring axons. OPCs inside the six axon/glia compartments were treated with a high concentration of ceramide (150 ?M) to confirm the fluidic isolation among the satellite compartments. In addition, isolated axons were treated with varying concentrations of chondroitin sulfate proteoglycan (CSPG, 0-25 ?g ml(-1)) within a single device to demonstrate the parallel localized biomolecular treatment capability of the device. These results indicate that the proposed platform can be used as a powerful tool to study CNS axonal biology and axon-glia interactions with the capacity for localized biomolecular treatments.
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Project description:Signaling through cyclic AMP (cAMP) has been implicated in the regulation of Schwann cell (SC) proliferation and differentiation. In quiescent SCs, elevation of cAMP promotes the expression of proteins associated with myelination such as Krox-20 and P0, and downregulation of markers associated with the non-myelinating SC phenotype. We have previously shown that the motor protein myosin II is required for the establishment of normal SC-axon interactions, differentiation and myelination, however, the mechanisms behind these effects are unknown. Here we report that the levels and activity of myosin light chain kinase (MLCK), an enzyme that regulates MLC phosphorylation in non-muscle cells, are dramatically downregulated in SCs after cAMP treatment, in a similar pattern to that of c-Jun, a known inhibitor of myelination. Knockdown of MLCK in SCs mimics the effect of cAMP elevation, inducing plasma membrane expansion and expression of Krox-20 and myelin proteins. Despite activation of myelin gene transcription these cells fail to make compact myelin when placed in contact with axons. Our data indicate that myosin II activity is differentially regulated at various stages during myelination and that in the absence of MLCK the processes of SC differentiation and compact myelin assembly are uncoupled.
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Project description:Rapid saltatory nerve conduction is facilitated by myelin structure, which is composed of Schwann cells in the peripheral nervous system. Schwann cells drastically change their phenotype following peripheral nerve injury. These phenotypic changes are required for efficient degeneration/regeneration. We previously identified ZNRF1 as an E3 ubiquitin ligase containing a RING finger motif, whose expression is upregulated in the Schwann cells following nerve injury. This suggested that posttranscriptional regulation of protein expression in Schwann cells may be involved in their phenotypic changes during nerve degeneration/regeneration. Here we report the identification of glutamine synthetase (GS), an enzyme that synthesizes glutamine using glutamate and ammonia, as a substrate for E3 activity of ZNRF1 in Schwann cells. GS is known to be highly expressed in differentiated Schwann cells, but its functional significance has remained unclear. We found that during nerve degeneration/regeneration, GS expression is controlled mostly by ZNRF1-dependent proteasomal degradation. We also found that Schwann cells increase oxidative stress upon initiation of nerve degeneration, which promotes carbonylation and subsequent degradation of GS. Surprisingly, we discovered that GS expression regulates Schwann cell differentiation; i.e., increased GS expression promotes myelination via its enzymatic activity. Among the substrates and products of GS, increased glutamate concentration inhibited myelination and yet promoted Schwann cell proliferation by activating metabotropic glutamate receptor signaling. This would suggest that GS may exert its effect on Schwann cell differentiation by regulating glutamate concentration. These results indicate that the ZNRF1-GS system may play an important role in correlating Schwann cell metabolism with its differentiation.
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Project description:The capacity of milli and micro litre bioreactors to accelerate process development has been successfully demonstrated in traditional biotechnology. However, for regenerative medicine present smaller scale culture methods cannot cope with the wide range of processing variables that need to be evaluated. Existing microfabricated culture devices, which could test different culture variables with a minimum amount of resources (e.g. expensive culture medium), are typically not designed with process development in mind. We present a novel, autoclavable, and microfabricated scale-down device designed for regenerative medicine process development. The microfabricated device contains a re-sealable culture chamber that facilitates use of standard culture protocols, creating a link with traditional small-scale culture devices for validation and scale-up studies. Further, the modular design can easily accommodate investigation of different culture substrate/extra-cellular matrix combinations. Inactivated mouse embryonic fibroblasts (iMEF) and human embryonic stem cell (hESC) colonies were successfully seeded on gelatine-coated tissue culture polystyrene (TC-PS) using standard static seeding protocols. The microfluidic chip included in the device offers precise and accurate control over the culture medium flow rate and resulting shear stresses in the device. Cells were cultured for two days with media perfused at 300 µl.h(-1) resulting in a modelled shear stress of 1.1×10(-4) Pa. Following perfusion, hESC colonies stained positively for different pluripotency markers and retained an undifferentiated morphology. An image processing algorithm was developed which permits quantification of co-cultured colony-forming cells from phase contrast microscope images. hESC colony sizes were quantified against the background of the feeder cells (iMEF) in less than 45 seconds for high-resolution images, which will permit real-time monitoring of culture progress in future experiments. The presented device is a first step to harness the advantages of microfluidics for regenerative medicine process development.
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Project description:Adipose-derived stem cells (ADSCs) can differentiate into Schwann cells (SCs) at the site of nerve injury, where Schwann cell-derived exosomes (SC-Exos) are suspected to exert an induction effect. Our study aimed to induce the differentiation of ADSCs in vitro using SC-Exos and to investigate the mechanisms involved through miRNA sequencing. Subcutaneous fat was used to extract ADSCs. Exosomes were extracted from Schwann cell lines (RSC96) using ultracentrifugation and were able to be taken up by human ADSCs. After 8 days of induction of ADSCs by SC-Exos, phenotypic characteristics were observed by examining the expression of SC markers (S100ß, NGFR, MPZ, GFAP) through RT-qPCR, Western blot and immunofluorescence. The RNA and protein expression levels of S100ß, NGFR, MPZ, and GFAP were found to be significantly higher in the SC-Exo induction group than in the uninduced group, which was also consistent with the immunofluorescence results. Additionally, miRNA sequencing was performed on exosome-induced ADSCs, followed by bioinformatic analysis and validation of the results. According to the sequencing results, there were a total of 94 differentially expressed miRNAs. Bioinformatics analysis indicated that 3506 Gene Ontology terms and 98 Kyoto Encyclopedia of Genes and Genomes pathways were significantly enriched. Ten miRNAs, 5 target mRNAs and elevated expression of the PIK3CD/Akt pathway were validated by RT-qPCR or Western blot, which is consistent with the sequencing results. Our study demonstrates that the utility of SC-Exos is effective in inducing the differentiation of ADSCs into SCs, in which these validated differentially expressed miRNAs exert a vital effect. This work provides a new paradigm via rationally applying Schwann cell-derived exosomes as a promising therapeutic option for repairing peripheral nerve injury.
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Project description:Salt removal is a prerequisite for electrospray ionization mass spectrometry (ESI-MS) analysis of biological samples. Rapid desalting and a low volume connection to an electrospray tip are required for time-resolved measurements. We have developed a microfabricated desalting device that meets both requirements, thus providing the foundational technology piece for transient ESI-MS measurements of complex biological liquid specimens. In the microfabricated device, the sample flows in a channel separated from a higher flow rate, salt-free counter solution by a monolithically integrated nanoporous alumina membrane, which can support pressure differences between the flow channels of over 600 kPa. Salt is removed by exploiting the large difference in diffusivities between salts and the typical ESI-MS target bioanalytes, e.g., peptides and proteins. We demonstrate the capability to remove 95% of salt from a sample solution in ?1 s while retaining sufficiently high concentration of a relatively low molecular weight protein, cytochrome-c, for ESI-MS detection.
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Project description:The remarkable plasticity of Schwann cells (SCs) is essential for nerve regeneration but also contributes to neuropathies and cancer progression. It has not yet been investigated whether the adaptive potential of SCs is manifested in stromal, tumor associated SCs characteristically found within a benign subtype of neuroblastic tumors (NBTs). We here performed transcriptome profiling of human NBTs, rich and poor in SC stroma, as well as human injured nerves, rich in repair SCs, revealing that stromal SCs exhibit a repair SC characteristic gene expression signature. In turn, primary repair SCs had a pro-differentiating and anti-proliferative effect on NBT cell lines after direct and trans-well co-culture. Within the pool of secreted stromal/repair SC factors, we identified EGFL8, a matricellular protein with so far undescribed function, to induce neuronal differentiation of aggressive NBT cells. EGFL8 expression further correlated with favorable tumor stage and increased patient survival. Our findings suggest that stromal SCs exert nerve repair associated functions in the tumor-environment and underline the therapeutic value of SC-derived factors for aggressive, SC stroma-poor NBTs.
2021-01-13 | PXD018267 | Pride
Project description:The data is related to the research article entitled "Arf6 mediates Schwann cell differentiation and myelination" [1]. To further investigate the role of Arf6 in promoting myelination by Schwann cells in vivo, we have characterized an another line (#2) of small-hairpin (sh)RNA transgenic mice targeting Arf6. The number of transgenes per one allele in this line was very low (2 transgenes), comparing with high copies in the previous line (#1, 20 transgenes) [1]. In 4 days of neonatal age, transgenic mice exhibited decreased myelin thickness; however, decreased levels were not as much as those in the line #1, likely depending on transgene copy number. In 60-day-old mice, the difference became smaller. On the other hand, transgene?s effect was not related to cell proliferation and apoptosis. These data support the key role of Arf6 in Schwann cell myelination, especially in the initiation.
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Project description:Rhizomelic chondrodysplasia punctata (RCDP) is a developmental disorder characterized by hypotonia, cataracts, abnormal ossification, impaired motor development, and intellectual disability. The underlying etiology of RCDP is a deficiency in the biosynthesis of ether phospholipids, of which plasmalogens are the most abundant form in nervous tissue and myelin; however, the role of plasmalogens in the peripheral nervous system is poorly defined. Here, we used mouse models of RCDP and analyzed the consequence of plasmalogen deficiency in peripheral nerves. We determined that plasmalogens are crucial for Schwann cell development and differentiation and that plasmalogen defects impaired radial sorting, myelination, and myelin structure. Plasmalogen insufficiency resulted in defective protein kinase B (AKT) phosphorylation and subsequent signaling, causing overt activation of glycogen synthase kinase 3β (GSK3β) in nerves of mutant mice. Treatment with GSK3β inhibitors, lithium, or 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8) restored Schwann cell defects, effectively bypassing plasmalogen deficiency. Our results demonstrate the requirement of plasmalogens for the correct and timely differentiation of Schwann cells and for the process of myelination. In addition, these studies identify a mechanism by which the lack of a membrane phospholipid causes neuropathology, implicating plasmalogens as regulators of membrane and cell signaling.
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