Purification of Exosomes from Primary Schwann Cells, RNA Extraction, and Next-Generation Sequencing of Exosomal RNAs

Gregorio, Cristian De; Diaz, Paula; López-Leal, Rodrigo; Manque, Patrício; Court, Felipe A.

doi:10.1007/978-1-4939-7649-2_19

Cited by 20 publications

(22 citation statements)

References 19 publications

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“…Exosomes containing proteins, RNA, lipids, and metabolites, can be engulfed by local tissues or released into body fluid to affect distant target organs, and exosomes are critical in intercellular communication in physiological and pathological conditions (9). It is believed that M2 macrophage-derived exosomes (M2-exos) transfer certain proteins or signals to tumor cells to regulate their migration (10).…”

Section: Introductionmentioning

confidence: 99%

RETRACTED: Mechanisms of M2 Macrophage-Derived Exosomal Long Non-coding RNA PVT1 in Regulating Th17 Cell Response in Experimental Autoimmune Encephalomyelitisa

Xia

et al. 2020

Front. Immunol.

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Long non-coding RNA (lncRNA) is pivotal for multiple sclerosis (MS), but the potential mechanism of lncRNA PVT1 in MS animal model, experimental autoimmune encephalomyelitis (EAE) still remains unclear. In this study, macrophages were firstly isolated and induced to polarize into M2 macrophages. M2 macrophage-derived exosomes (M2-exos) were extracted and identified, and EAE mouse model was established and treated with M2-exos. The effect of M2-exos on EAE mice was evaluated by clinical scores. The proportion of Treg and Th17 cells in spinal cord cells and splenocytes, and levels of inflammatory factors were measured. The targeting relationships among PVT1, miR-21-5p, and SOCS5 were verified. The expression of JAKs/STAT3 pathway-related proteins was measured. After M2-exo treatment, the clinical score of EAE mice decreased, and demyelination and inflammatory infiltration improved; Th17 cells decreased, Treg cells increased, and the levels of inflammatory factors decreased significantly. SOCS5 and PVT1 were downregulated and miR-21-5p was upregulated in EAE mice. PVT1 could sponge miR-21-5p to regulate SOCS5. SOCS5 alleviated EAE symptoms by repressing the JAKs/STAT3 pathway. Together, M2-exos-carried lncRNA PVT1 sponged miR-21-5p to upregulate SOCS5 and inactivate the JAKs/STAT3 pathway, thus reducing inflammation and protecting EAE mice. This study may offer novel treatments for MS.

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Section: Introductionmentioning

confidence: 99%

RETRACTED: Mechanisms of M2 Macrophage-Derived Exosomal Long Non-coding RNA PVT1 in Regulating Th17 Cell Response in Experimental Autoimmune Encephalomyelitisa

Xia

et al. 2020

Front. Immunol.

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“…EVs were isolated and purified from the primary SC and MS-SC cultures following previously established protocol 33 , 34 . EVs were harvested from passage 2-3 primary SCs.…”

Section: Methodsmentioning

confidence: 99%

“…EVs were isolated from SC and MS-SC cultures, as previously described 33 , 34 . Total RNA extracted from EVs was used for miRNA sequencing.…”

Section: Methodsmentioning

confidence: 99%

Mechanical stimulation of Schwann cells promote peripheral nerve regeneration via extracellular vesicle-mediated transfer of microRNA 23b-3p

Xia¹,

Gao²,

Li³

et al. 2020

Theranostics

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Rationale: Peripheral nerves are unique in their remarkable elasticity. Schwann cells (SCs), important components of the peripheral nervous system (PNS), are constantly subjected to physiological and mechanical stresses from dynamic stretching and compression forces during movement. So far, it is not clear if SCs sense and respond to mechanical signals. It is also unknown whether mechanical stimuli can interfere with the intercellular communications between neurons and SCs, and what role extracellular vesicles (EVs) play in this process. The present study aimed to examine the effect of mechanical stimuli on the EV-mediated intercellular communication between neurons and SCs, explore their effect on axonal regeneration, and investigate the underlying mechanism. Methods: Purified SCs were stimulated using a magnetic force-based mechanical stimulation (MS) system and EVs were purified from mechanically stimulated SCs (MS-SCs-EVs) and non-stimulated SCs (SCs-EVs). The effect of MS-SCs-EVs on axonal elongation was examined in vitro and in vivo . High throughput miRNA sequencing was performed to compare the differential miRNA profiles between MS-SCs-EVs and SCs-EVs. The functional role of differentially expressed miRNAs on neurite extension in MS-SCs-EVs was examined. Also, the putative target genes of differentially expressed miRNAs in MS-SCs-EVs were predicted by bioinformatics tools, and the regulatory effect of those miRNAs on putative target genes was validated both in vitro and in vivo . Results: The MS-SCs-EVs showed an average size of 137.52±1.77 nm, and could be internalized by dorsal root ganglion (DRG) neurons. Compared to SCs-EVs, MS-SCs-EVs showed a stronger ability to enhance neurite outgrowth in vitro and nerve regeneration in vivo . High throughput miRNA sequencing identified a number of differentially expressed miRNAs in MS-SCs-EVs. Further analysis of those EV-miRNAs demonstrated that miR-23b-3p played a predominant role in MS-SCs-EVs since its deprivation abolished their enhanced axonal elongation. Furthermore, we identified neuropilin 1 (Nrp1) in neurons as the target gene of miR-23b-3p in MS-SCs-EVs. This observation was supported by the evidence that miR-23b-3p could decrease Nrp1-3'-UTR-WT luciferase activity in vitro and down-regulate Nrp1 expression in neurons. Conclusion: Our findings suggested that mechanical stimuli are capable of modulating the intercellular communication between neurons and SCs by altering miRNA composition in MS-SCs-EVs. Transfer of miR-23b-3p by MS-SCs-EVs from mechanically stimulated SCs to neurons decreased neuronal Nrp1 expression, which was responsible, at least in part, for the beneficial effect of MS-SCs-EVs on axonal regeneration. Our results highlighted the potential therapeutic value of MS-SCs-EVs and miR-23b-3p-en...

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“…SCs were obtained from newborn P2-P3 SD rat sciatic nerves as previously described (Lopez-Verrilli et al, 2013;De Gregorio et al, 2018), expanded over laminin coating and maintained on DMEM with 10% FBS (Gibco Life Technologies, cat. #10437028) supplemented with 2 µM forskolin (Sigma, cat.…”

Section: Schwann Cell Culturementioning

confidence: 99%

Schwann cell reprogramming into repair cells increases miRNA-21 expression in exosomes promoting axonal growth

López-Leal

Díaz-Viraqué

Catalán

et al. 2020

Journal of Cell Science

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Functional recovery after peripheral nerve damage is dependent on the reprogramming of differentiated Schwann cells (dSCs) into repair Schwann cells (rSCs), which promotes axonal regeneration and tissue homeostasis. Transition into a repair phenotype requires expression of c-Jun and Sox2, which transcriptionally mediates inhibition of the dSC program of myelination and activates a non-cellautonomous repair program, characterized by the secretion of neuronal survival and regenerative molecules, formation of a cellular scaffold to guide regenerating axons and activation of an innate immune response. Moreover, rSCs release exosomes that are internalized by peripheral neurons, promoting axonal regeneration. Here, we demonstrate that reprogramming of Schwann cells (SCs) is accompanied by a shift in the capacity of their secreted exosomes to promote neurite growth, which is dependent on the expression of c-Jun (also known as Jun) and Sox2 by rSCs. Furthermore, increased expression of miRNA-21 is responsible for the pro-regenerative capacity of rSC exosomes, which is associated with PTEN downregulation and PI3-kinase activation in neurons. We propose that modification of exosomal cargo constitutes another important feature of the repair program of SCs, contributing to axonal regeneration and functional recovery after nerve injury.

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Purification of Exosomes from Primary Schwann Cells, RNA Extraction, and Next-Generation Sequencing of Exosomal RNAs

Cited by 20 publications

References 19 publications

RETRACTED: Mechanisms of M2 Macrophage-Derived Exosomal Long Non-coding RNA PVT1 in Regulating Th17 Cell Response in Experimental Autoimmune Encephalomyelitisa

RETRACTED: Mechanisms of M2 Macrophage-Derived Exosomal Long Non-coding RNA PVT1 in Regulating Th17 Cell Response in Experimental Autoimmune Encephalomyelitisa

Mechanical stimulation of Schwann cells promote peripheral nerve regeneration via extracellular vesicle-mediated transfer of microRNA 23b-3p

Schwann cell reprogramming into repair cells increases miRNA-21 expression in exosomes promoting axonal growth

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