The Immortalization And Differentiation Of Human Urine-derived Stem Cells

Objective: piggyBac system can start screening in stem cellsefficiently promoter gene expression, and applied to simian virus40largeT antigen expression (SV40Tag) gene, the establishment of immortalizedhuman urine-derived stem cells (USC). Immortalized by biologicalidentification of urinary-derived stem cells, stem cell differentiation toclarify its potential and induced to differentiate by bone morphogeneticprotein (BMP9), observation of urine-derived stem cells osteogenicactivity and identified, further analysis BMP9induced osteogenicmechanisms for tissue engineering and regenerative medicine provide astable source of cells.Method: Construct and compare with different promoter plasmidswith a red fluorescent protein (RFP) and firefly luciferase (Fluc) as areporter gene, the establishment of stable expression of stem cell lines byliposome transfection methods. By RFP expression intensity, Flucquantitative detection and flow cytometry, screening stable promoterhighly expressed in stem cells. With this promoter band SV40Tagconstruct plasmid gene expression, will SV40Tag transposase gene plasmid and a plasmid were transfected into clinical isolates of humanurine-derived stem cells. Hygromycin after screening positive clones andserially passaged to observe the biological characteristics of cellmorphology and proliferation, RT-PCR, immunofluorescence detection oftransfected cells, the cells subcutaneously in nude mice to detect theirsafety. Use BMP9induced osteogenic differentiation in vitro cell alkalinephosphatase (ALP) staining, Alizarin S staining, Oil red O, Micro Massculture and other specific staining to detect the osteogenic and adipogenicdifferentiation of cartilage. In vivo experiments further use of Micro-CT,biopsy staining urine-derived stem cell osteogenic differentiation in BMP9induced.Results: RFP fluorescent display, Fluc and flow quantitative test,hEFH promoter expression in stem cell lines exogenous gene was strongerthan the other promoters, using hEFH start SV40Tag gene expression inthe urine-derived stem cells, obtained by screening positive clonesexpanding culture, that immortalized the urine-derived stem cells (iUSC),proliferative ability, continuous subculture. RT-PCR confirmed iUSCSV40Tag gene expression, but with the Flippers recombination enzyme(Flp) can handle iUSC gene knockout RFT-SV40Tag be reversedimmortalized. Immunofluorescence staining was detected in the cell linesexpress stem cell surface markers CD73, CD44, CD90(Thy-1), CD29(Integrin), CD105(Endoglin), CD166(ALCAM), BMPR II, SSEA4, CD117, CD133. Application BMP9induced iUSC differentiation, cellalkaline phosphatase staining, Alizarin red staining, oil red stainingshowed that the cells into osteoblasts, chondrocytes and adipocytestransfected cells were inoculated subcutaneously and muscle, four weeksafter no tumors formed by Micro-CT and pathological staining showed thatthe cells BMP9induced in vivo has a good portion of cartilage andosteogenic differentiation into fat.Conclusion: Conclusion: The use of piggyBac system successfullyscreened suitable for gene expression of stem cell promoter hEFH, andsuccessfully build SV40Tag immortalized in urine-derived stem cells withthe promoter by liposome transfection techniques, biological identificationof the cells with mesenchymal stem cell characteristics using BMP9induced to differentiate in vitro and in vivo experiments have confirmedthat the cells have osteogenic, adipogenic, chondrogenic differentiation,further confirmed the multi-differentiation potential of immortalized humanurine-derived stem cells can be induced to differentiate further for thegenitourinary system the damage repair, and provides a safe and stable cellsource for tissue engineering and regeneration in vitro studies and thedevelopment of medical applications.