Abstract
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.
Highlights
Peripheral nerve injury (PNI) is one of the common complications of various types of trauma and mainly occurs after motor vehicle accidents, natural disasters, work-related accidents and sports injuries (Wang et al, 2019)
The possible mechanisms have been confirmed by subsequent studies to exist in two aspects: transplanted mesenchymal stem cells (MSCs) can secrete various types of neurotrophic factors, such as brainderived neurotrophic factor (BDNF), nerve growth factor (NGF), glial growth-like factor (GGF), and neuregulin-1 (NRG-1), to promote nerve repair (Jiang et al, 2017; Lavorato et al, 2021); transplanted MSCs are stimulated to differentiate into Schwann cells (SCs) in the nerve injury area in vivo (Chen et al, 2006; Ladak et al, 2011), and Schwann cells can provide growth channels for axonal regeneration by forming Büngner bands, so that axons can continue to grow across the defect area
Detection of exosomal markers by Western blot revealed that CD9, CD63, and CD81 were positive, while nonexosomal markers, such as GM130, were negatively expressed, and GAPDH were weakly expressed relative to Adipose-derived stem cells (ADSCs) (Figure 2C)
Summary
Peripheral nerve injury (PNI) is one of the common complications of various types of trauma and mainly occurs after motor vehicle accidents, natural disasters, work-related accidents and sports injuries (Wang et al, 2019). Many in vivo experiments in animals have shown that transplantation of MSCs can promote injured peripheral nerve regeneration. The possible mechanisms have been confirmed by subsequent studies to exist in two aspects: transplanted MSCs can secrete various types of neurotrophic factors, such as brainderived neurotrophic factor (BDNF), nerve growth factor (NGF), glial growth-like factor (GGF), and neuregulin-1 (NRG-1), to promote nerve repair (Jiang et al, 2017; Lavorato et al, 2021); transplanted MSCs are stimulated to differentiate into Schwann cells (SCs) in the nerve injury area in vivo (Chen et al, 2006; Ladak et al, 2011), and Schwann cells can provide growth channels for axonal regeneration by forming Büngner bands, so that axons can continue to grow across the defect area. The specific reasons and mechanisms for inducing MSCs to differentiate into Schwann cells are still unclear
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