Objective:To compare the expression levels of pluripotent genes among incomplete reprogrammed colonies and induced pluripotent stem cells(iPSCs),to explore the relationship between the expression of pluripotent genes ...Objective:To compare the expression levels of pluripotent genes among incomplete reprogrammed colonies and induced pluripotent stem cells(iPSCs),to explore the relationship between the expression of pluripotent genes and incomplete reprogramming.Methods:four genes(Oct4,Sox2,Klf4,C-Myc) were introduced into human foreskin fibroblasts(HFFs) by retroviruses.The HFFs were induced to reprogramming.Different forms of colonies were picked up,analyzed,and compared with iPSCs from different aspects,including the morphology of clones,alkaline phosphatase(AP) staining,immuno-fluorescence,and Q-PCR.Results:In the reprogramming process,different colonies were emerged,some of them exhibited typical human embryonic stem cell morphology(eg.,compact colonies,high nucleus-to-cytoplasm ratios,and prominent nucleoli).However,these colonies couldn't maintain these characters after passage.There was an intermediate state,named partially reprogramming.Through analysis and identification,AP staining results were weakly positive,compared with iPSC colonies.The immuno-fluorescence staining demonstrated these colonies just expressed pluripotent protein Oct4.Q-PCR indicated that the expression of exogenous transcription factors was inappropriate,either at a high level or at a low level.Most of the endogenous pluripotency genes were expressed at a low level.Conclusions:It may be one of the causes of incomplete reprogramming that the exogenous pluripotent gene is low-expressed or over-expressed,and successful reprogramming may depend on a specific stoichiometric balance of Oct4,Sox2,Klf4 and c-Myc.展开更多
基金The 973 program of Ministry of Science and Technology of China(No.2012CB966502)The Key International Cooperation Program of Science and Technology Department of Hainan Province(No.GJXM201106,KJHZ2014-11)MOST of China(No.2014DFA30180)
文摘Objective:To compare the expression levels of pluripotent genes among incomplete reprogrammed colonies and induced pluripotent stem cells(iPSCs),to explore the relationship between the expression of pluripotent genes and incomplete reprogramming.Methods:four genes(Oct4,Sox2,Klf4,C-Myc) were introduced into human foreskin fibroblasts(HFFs) by retroviruses.The HFFs were induced to reprogramming.Different forms of colonies were picked up,analyzed,and compared with iPSCs from different aspects,including the morphology of clones,alkaline phosphatase(AP) staining,immuno-fluorescence,and Q-PCR.Results:In the reprogramming process,different colonies were emerged,some of them exhibited typical human embryonic stem cell morphology(eg.,compact colonies,high nucleus-to-cytoplasm ratios,and prominent nucleoli).However,these colonies couldn't maintain these characters after passage.There was an intermediate state,named partially reprogramming.Through analysis and identification,AP staining results were weakly positive,compared with iPSC colonies.The immuno-fluorescence staining demonstrated these colonies just expressed pluripotent protein Oct4.Q-PCR indicated that the expression of exogenous transcription factors was inappropriate,either at a high level or at a low level.Most of the endogenous pluripotency genes were expressed at a low level.Conclusions:It may be one of the causes of incomplete reprogramming that the exogenous pluripotent gene is low-expressed or over-expressed,and successful reprogramming may depend on a specific stoichiometric balance of Oct4,Sox2,Klf4 and c-Myc.
