Persistent Stemnesss Of HESCs And Notch1Expression Changes In A Self-made Serum-free Medium

Objective：Observing the stemness of hESCs and change trend of notch1expression in aself-made serum-free medium, in order to review the capability of this medium toeffectively expand hESCs in vitro, and probe the the role of activated notch pathwayin the maintenance of stemness in these cells.Methods：Culturing hESCs in the self-made serum-free medium, deriving the20th passageand30th passage cells; Detecting the expression of pluripotency markers such asOCT4,Nanog and SSEA-4in these cells by immunofluorescence staining technics;Observing alkaline phosphatase activity of these cells; Inoculatingimmunocompromised mices with hESCs, observing the formation of endoderm,neuroectoderm and mesoderm in generated teraomas from immunocompromisedmices; Measuring the changes of notch1expression in these cells by real-time PCRand western-bolt, analysising datas.Results：(1) Immunofluorescence staining: hESCs express OCT-4,Nanog and SSEA-4in both passages of hESCs;(2) Alkaline phosphatase staining: hESCs are stained blue, and with a strongpositive reaction in both passages of hESCs.(3) Staining tissue sections of teratomas produced by hESCs: histopathologicalanalysis of teratomas reveale multiple tissue types including endoderm,neuroectoderm and mesoderm.(4)Expression of notch1genes in these cells: the expression levels of Notch1at30th passage hESCs is8.4±0.23folds more than that at20th passage hESCs.(5) Expression levels of notch1protein in these cells: The relative expression of300kd fragment is0.17±0.013at30th passage hESCs, and0.016±0.002at20thpassage. Farthermore, the relative expression of120kd fragment is0.41±0.021at30th passage hESCs, and0.36±0.018at20th passage. So, The expression of notch1proteinat30th passage hESCs is significantly highly than that at20th passage(p<0.05).Conclusion：(1)HESCs can persistently proliferate in our self-made serum-free medium, andmaintain its ability to differentiate into cells lineages and infinitely self-renew.(2) HESCs steadily express high levels of notch1, which implys our self-madeserum-free medium may depend upon the activated notch pathwayto propagatehESCs in vitro.