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Human Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles Carrying MicroRNA-181c-5p Promote BMP2-Induced Repair of Cartilage Injury through Inhibition of SMAD7 Expression.

ABSTRACT: The therapy role of mesenchymal stem cell- (MSC-) derived extracellular vesicles (EVs) in cartilage regeneration has been well studied. Herein, we tried to analyze the role of human umbilical cord MSC- (hUCMSC-) EVs carrying microRNA- (miR-) 181c-5p in repair of cartilage injury. After successful isolation of hUCMSCs, the multidirectional differentiation abilities were analyzed. Then, the EVs were isolated and identified. After coculture of PKH26-labled EVs with bone marrow MSCs (BMSCs), the biological behaviors of which were detected. The relationship between the predicted early posttraumatic osteoarthritis-associated miRNA, miR-181c-5p, and SMAD7 was verified. Gain- and loss-of functions were performed for investing the role of miR-181c-5p and SMAD7 in BMP-induced chondrogenesis in vitro and in vivo. hUCMSC-EVs could be internalized by BMSCs and promote the proliferative, migratory, and chondrogenic differentiation potentials of BMSCs. Additionally, miR-181c-5p could target and inhibit SMAD7 expression to promote the bone morphogenic protein 2- (BMP2-) induced proliferative, migratory, and chondrogenic differentiation potentials of BMSCs. Also, overexpression of SMAD7 inhibited the repairing effect of BMP2, and overexpression of BMP2 and miR-181c-5p further promoted the repair of cartilage injury in vivo. Our present study highlighted the repairing effect of hUCMSC-EVs carrying miR-181c-5p on cartilage injury.

SUBMITTER: Zhang Q

PROVIDER: S-EPMC9249498 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Generating insulin-producing cells (IPCs) from human pluripotent stem cells is a promising method for studying the molecular mechanism underlying pancreas development and a potential treatment source for type 1 diabetes. Previous studies have shown that miR-181c-5p is highly enriched in adult islets; however, its role in pancreatic β cell differentiation is poorly understood. In this study, we differentiated human induced pluripotent stem cells (hiPSCs) into IPCs in a stepwise process that recapitulated pancreas organogenesis and observed that miR-181c-5p continuously accumulated throughout the entire differentiation process. hiPSCs were transduced with lentiviral vectors containing human miR-181c-5p precursor, which significantly increased the endodermal markers SOX17, FOXA2, CXCR4 and GATA4 and pancreatic endocrine-specific gene expression, including PDX1, NKX6.1, MAFA and Insulin. miR-181c-5p overexpression exerted little effect on the efficiency of definitive endoderm, whereas it promoted the differentiation of pancreatic progenitors and IPCs, especially for NKX6.1-positive and insulin-positive cells differentiation. Transplanted these cells exhibit glucose-stimulated C-peptide secretion in vivo and protect mice from chemically induced diabetes. It was found that miR-181c-5p directly targets the 3'UTR of smad7 and TGIF2 mRNA, which are known to be endogenous repressors of TGF-β-smad2/3 signaling, to decrease their mRNA and protein levels. Furthermore, overexpressed miR-181c-5p led to an elevation of the smad2/3 phosphorylation levels in hiPSC-derived cells, while treatment with smad2/3 inhibitors following miR-181c-5p overexpression had opposite effects on IPC formation. These results suggest that miR-181c-5p is critically involved in pancreatic lineage commitment through direct repression of smad7 and TGIF2 and that it modulates TGF-β-smad2/3 signaling activation and increases the feasibility of using patient-specific hiPSCs for β cell replacement therapy for type 1 diabetes.

Project description:BackgroundSpinal cord injury (SCI) is an inflammatory condition, and excessive adenosine triphosphate (ATP) is released into the extracellular space, which can be catabolized into adenosine by CD73. Extracellular vesicles have been designed as nano drug carriers in many diseases. However, their impacts on delivery of CD73 after SCI are not yet known. We aimed to construct CD73 modified extracellular vesicles and explore the anti-inflammatory effects after SCI.MethodsCD73 engineered extracellular vesicles (CD73+ hucMSC-EVs) were firstly established, which were derived from human umbilical cord mesenchymal stem cells (hucMSCs) transduced by lentiviral vectors to upregulate the expression of CD73. Effects of CD73+ hucMSC-EVs on hydrolyzing ATP into adenosine were detected. The polarization of M2/M1 was verified by immunofluorescence. Furthermore, A2aR and A2bR inhibitors and A2bR knockdown cells were used to investigate the activated adenosine receptor. Biomarkers of microglia and levels of cAMP/PKA were also detected. Repetitively in vivo study, morphology staining, flow cytometry, cytokine analysis, and ELISA assay, were also applied for verifications.ResultsCD73+ hucMSC-EVs reduced concentration of ATP and promoted the level of adenosine. In vitro experiments, CD73+ hucMSC-EVs increased macrophages/microglia M2:M1 polarization, activated adenosine 2b receptor (A2bR), and then promoted cAMP/PKA signaling pathway. In mice using model of thoracic spinal cord contusion injury, CD73+ hucMSC-EVs improved the functional recovery after SCI through decreasing the content of ATP in cerebrospinal fluid and improving the polarization from M1 to M2 phenotype. Thus, the cascaded pro-inflammatory cytokines were downregulated, such as TNF-α, IL-1β, and IL-6, while the anti-inflammatory cytokines were upregulated, such as IL-10 and IL-4.ConclusionsCD73+ hucMSC-EVs ameliorated inflammation after spinal cord injury by reducing extracellular ATP, promoting A2bR/cAMP/PKA pathway and M2/M1 polarization. CD73+ hucMSC-EVs might be promising nano drugs for clinical application in SCI therapy.

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Human Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles Carrying MicroRNA-181c-5p Promote BMP2-Induced Repair of Cartilage Injury through Inhibition of SMAD7 Expression.

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