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Lentivirus transduced interleukin-1 receptor antagonist gene expression in murine bone marrow-derived mesenchymal stem cells in vitro.

ABSTRACT: Genetically modified mesenchymal stem cells have been used in attempts to increase the expression of interleukin?1 receptor antagonist (IL?1Ra); however, the attempts thus far have been unsuccessful. The aim of the present study was to investigate whether the lentivirus transduced IL?1Ra gene was able to be stably expressed in murine bone marrow?derived mesenchymal stem cells (mBMSCs) in vitro. In the present study, third generation lentiviral (Lv) vectors transducing the IL?1Ra/green fluorescent protein (copGFP) gene were constructed and transfected into mBMSCs to establish the Lv.IL?1Ra.copGFP/mBMSCs, which were evaluated using fluorescence microscopy, flow cytometry, cell viability analysis using a cell counting kit?8 kit, Trypan blue staining and an MTT growth kinetics assay. The expression of IL?1Ra was analyzed using reverse transcription-quantitative polymerase chain reaction and western blotting. The results demonstrated that the Lv.IL?1Ra/copGFP vector was successfully constructed. The mBMSCs exhibited a short proliferation life, however they had good growth kinetics at an early stage and improved viability following efficient transduction of the IL?1Ra gene. IL?1Ra was overexpressed following transfection of mBMSCs. In conclusion, lentiviral vector transduced mBMSCs were able to efficiently express exogenous Il?1Ra under certain conditions and had a marked capacity for proliferation.

SUBMITTER: He T

PROVIDER: S-EPMC4526042 | biostudies-literature | 2015 Sep

REPOSITORIES: biostudies-literature

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Lentivirus transduced interleukin-1 receptor antagonist gene expression in murine bone marrow-derived mesenchymal stem cells in vitro.

He Tao T Chi Guanghao G Tian Bo B Tang Tingting T Dai Kerong K

Molecular medicine reports 20150626 3

Genetically modified mesenchymal stem cells have been used in attempts to increase the expression of interleukin‑1 receptor antagonist (IL‑1Ra); however, the attempts thus far have been unsuccessful. The aim of the present study was to investigate whether the lentivirus transduced IL‑1Ra gene was able to be stably expressed in murine bone marrow‑derived mesenchymal stem cells (mBMSCs) in vitro. In the present study, third generation lentiviral (Lv) vectors transducing the IL‑1Ra/green fluorescen ...[more]

PMID: 26130370

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Project description:ObjectiveLubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs) are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1) whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2) whether aggregates of human MSCs promoted lubricin expression, and (3) whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats.MethodsFor in vitro analysis, human bone marrow (BM) MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages.ResultsIn in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4), which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in the superficial cartilage.ConclusionCartilage derived from MSCs expressed lubricin protein both in vitro and in vivo. Aggregation promoted lubricin expression of MSCs in vitro and transplantation of aggregates of MSCs regenerated cartilage including the superficial zone in a rat osteochondral defect model. Our results indicate that aggregated MSCs could be clinically relevant for therapeutic approaches to articular cartilage regeneration with an appropriate superficial zone in the future.

Dataset Information

Lentivirus transduced interleukin-1 receptor antagonist gene expression in murine bone marrow-derived mesenchymal stem cells in vitro.

Publications

Lentivirus transduced interleukin-1 receptor antagonist gene expression in murine bone marrow-derived mesenchymal stem cells in vitro.

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