Investigation Of Labeling Bone Marrow Mesenchymal Stem Cells And Their Differentiation Into Renal-Like Cells

Objective To establish a method of labeling bone marrow mesenchymal stem cell with PKH26 and DAPI in vitro and explore the biological activity of labeled MSC. To induce rat acute renal failure model by intra-muscle injection of 50% glycerol in SD rats and evaluate the therapeutic efficacy by injecting MSC intravenously and explore the possible mechanisms in vivo and in vitro.Methods Mesenchymal stem cell was stained with red fluorescent PKH26 and blue fluorescent DAPI. The growth and fluorescent intensity was observed by confocol laser microscopy and flow cytometer. The expression of nucleostemin, Bmi-1 genes in the cells was detected by RT-PCR. The characteristics of labeled MSC differentiating into osteoblasts in vitro were identified by ALP stain, Von kossa stain and bone morphogenetic protein3(BMP3) expression. Rat acute renal failure model was induced by intra-muscle injection of 50% glycerol in SD rats. MSC was injected intravenously into them. The therapeutic efficacy of MSC as well as the possible mechanisms about it were investigated by clinical biochemistry, molecular biology, immunohistochemical and pathological methods, fluorescence in situ hybridization(FISH) and so on. Rat MSC was cocultured with injured kidney by transwell in vitro, and whether the cells could differentiate into renal tubular-like epithelial cells was identified by confocol, RT-PCR, Nested PCR, real-time quantitative PCR.Results The labeled cells appeared red fluorescence. The intensity of fluorescence was related to the concentration of fluorochrome and the passages. The growth characteristic between the labeled and unlabeled cells was not different significantly. The expression of nucleostemin, Bmi-1 genes between them was not observed. After being induced, the cells appeared typical cell shape and biological characteristics of osteoblasts, positive for ALP stain, Von kossa stain and expressed BMP3 gene. Rat ARF model was established successfully. There were more exogenous MSC in injured kidney than in other organs such as heart, liver, lung and brain. The human 17αsatellite DNA sequence was amplified in rat heart, liver and different parts of rat kidney in the experiment for fetal MSC location. The exogenous cells from human MSC could be seen in rat kidney section by FISH analysis. The levels of serum creatinine and nitrogen decreased significantly in experimental groups than in control groups(P＜0.05). The kidney recovery situation in experimental groups was better than in control groups. After cocultured with injured kidney in vitro, the shape of rat MSC turned round, and they highly expressed cytokeratin 18(CK18) and aquaporin-1(AQP1).Conclusions MSC could be labeled with PKH26 and DAPI stably. The labeled cells still had the abilities of self-renewal and multi-differentiation. It was suggested that the labeling method could be applied to study the homing, plasticity and transplantation of MSC. Exogenous MSC could home to the injured kidney and cure rat ARF efficiently. The injured kidney tissue could induce rat MSC trans-differentiate into renal tubular-like epithelial ceils. These investigations could offer original experimental model and evidence about efficient cure of MSC to kidney disorders such as ARF, acute glomerulonephritis, and also open up a new path to them.