Study On Biological Property Changes Of Stem Cells From Fetus Cultured By Continued Culture In Vitro

Objective: To analyze the effects of in vitro continued culture on basicfeatures, oncogenicity and different iation capacity of stem cells from fetusincluding umbilical cord mesenchymal stem cell and human amnioticepithelial cell,and providing experimental basis for quality control of stemcellsMethods: Primarily cultured human amniotic epithelial cells （n=8）andmenchymal stem cells（n=3） were obtained from fetus tissues by using thesame separation protocol and were cultured in vitro. Cellular basic features（morphology， proliferation， cell cycle， apoptosis and senescence）,oncogenicity（the chromosome number and tumorigenic gene expression）and stem capacity （phenotype， stem gene expression and cardiac, neuraland hepatocyte differentiation ability）in passages7,14and21were evaluatedby means of CCK8, flow cytometry, real-time PCR,immunoblotting,immunofluorescence and cytochemical staining. On theother hand,cell phenotype, proliferation and the differentiation intocardiomyocyte-like cells of human amniotic epithelial cells were evaluatedwith continued culture in vitro,and we discussed the correlation with theSSEA-4expression and the differentiation into cardiomyocyte-like cells inhuman amniotic epithelial cells.Results:1. Study on biological property changes of cord mesenchymal stem cellscultured by continued culture in vitro1） Study on basic features of cord mesenchymal stem cells cultured by continued culture in vitroThe study of mesenchymal stem cells basic features indivated that MSCsgrew conglomerately with homogeneous fibroblastoid shapes du ringthe continuous passages,but the morphology were more bigger and the S andG2phase retardation phenomenon,but there were no significantly differencein passage7,14and21. The cell apoptosis ratio were no significantlydifference between different passages cells,and Annexin V-/PI-cell ratiowere more than90%.But senescence ratio（0.229±0.019）in passage21wasmore than passage7（0.032±0.018）,14（0.062±0.038）(P<0.05),and it wasonsistent with P21gene expression（P21：0.050±0.020，P14：0.007±0.001，P7：0.010±0.004）（P<0.05）. The experimental results showed that tumbilical cord mesenchymal stem cells in under passage14could maintainits basic features, senescence cell ratio was more higher in passage21.2） Study on oncogenicity of cord mesenchymal stem cells cultured bycontinued culture in vitroThe chromosome numbers were not changed b y chromosome analysis indifferent passages,and E-Ras oncogene expression were no significantlydifference between different passages cells， but the c-Myc gene expression（P7：0.014±0.006，P14：0.011±0.002，P21：0.047±0.005）was higherin passage21（P<0.05）. These results indicated that although cellchromosome numbers were normal, but some of oncogene expressionappeared obvious rise, suggesting higher algebra cells may have a highertumorigenicity.3) Study on stem character of cord mesenchymal stem cells cultured bycontinued culture in vitroThe surface marker including CD73,CD90and CD105positive ratio weremore than90%,and did not expression HLA-DR and CD45（P>0.05）. Ascell senescence ratio and oncogene expression were significantlyincreased,so we focused on the stem character and the cardiac, neuraldifferentiation ability in passage7and14.The experiment indicated thatOct3/4（P7：2.378E-5±1.347E-5，P14：1.955E-5±3.692E-6）、Nanog（P7：1.063E-5±5.722E-6，P14：6.517E-6±2.666E-6）、Vimentin （P7：0.507±0.144，P14：0.323±0.041），the ectoderm progenitor cell markersNestin（P7：9.421E-4±2.011E-4，P14：6.952E-4±2.906E-4）and mesodermal progenitor cell markers GATA4（P7：3.215E-5±3.716E-5，P14：5.082E-6±5.293E-6）expression decreased. endoderm progenitor cell markersAFP（P7：5.891E-5±2.439E-5，P14：9.916E-5±2.620E-5）expressionincreased. But there were no significantly difference（P>0.05）.The α-actininprotein expression in passages7（0.111±0.030）and14（0.033±0.006）was higher in passage7by immunoblotting after induction tocardiomyocyte-like cells（P<0.05）and suggested that the cells in passage7had high myocardial differentiation ability.The Nestin（27.2±12.0%）、β-tubulin Ⅲ（70.4±4.5%）positive cells ratio in passage14was higher thanin passage7by immunofluorescence （Nestin：20.9±0.8%；β-tubulinⅢ：38.6±10.2%） after induction to neural-like cells,and β-tubulin Ⅲ hadsignificantly difference（P<0.05）,which indicated that the cells in passage14had high neural-like cells differentiation ability. The Albumin geneexpression were higher in passage7（P7：16.433±12.921， P14：12.967±1.358）（P<0.05）and Albumin protein expression（P7：0.382±0.270，P14：0.260±0.127） were no significantly difference after induction to liver-likecells in early phase（P>0.05）.The results suggested that the cells maybe inpassage7had high liver-like differentiation ability,but there were biggerindividual differences2. Study on biological property changes of human amniotic epithelialcells cultured by continued culture in vitroThe SSEA-4positive cells in primarily cultured human amnioticepithelial cells from different fetal tissues were between26.7%-97%, whichindicated that there was great individual difference among amniotic t issuesamples. Moreover, with passage, the SSEA-4expression in human amnioticepithelial cells decreased significantly in passage2, which did not correlatewith the SSEA-4expression in primarily cultured human amniotic epithelialcells. In addition, the differentiation capacity of human amniotic epithelialcells into cardiomyocyte-like cells was also affected by individualdifference among different samples, which also did not correlate with theSSEA-4expression in primarily cultured human amniotic epith elial cells.Conclusion:1. Compared with umbilical cord mesenchymal stem cells cell inpassage21, the cell under passage14there was no obvious between senescence ratio and well maintained its basic characteristics, and ha d alower oncogene expression. and therefore more suitable for regenerativemedicine research and application.2. Differentiation experiments of umbilical cord mesenchymal stem cellsshow that, while under the influence of individual d ifferences. But continueto culture in vitro with different differentiation direction had differenteffect: lower myocardial and differentiation ability in low passages, cellswhile higher neural differentiation ability in high passages. Therefore weneeded to consider to chose fit cells in passages in the concrete applicationof in vitro to extend influence of umbilical cord mesenchymal stem cells todifferentiate between abilities, further strengthen the quality control of stemcells, improved the safety and effectiveness3.The SSEA-4positive cells in primarily cultured human amnioticepithelial cells from different fetal tissues indicated that there was greatindividual difference among amniotic tissue samples. We needed to establisha more accurate clinical samples screening index to express the stability forthe original generation high SSEA-4fetal samples, in order to realize tomonitor the quality of amniotic epithelial cells. In addition, SSEA-4expression level in vitro in the process of the inf luences of cultureconditions, continue to optimize culture conditions were needed to maintainits high expression,and the sample amniotic epithelial cells to myocardialcell differentiation samples the ability of the individual differences and theinfluence of culture conditions, further research was needed in the future.