Small Molecule Compounds Activated Expression Of Endogenous Pluripotent Genes In Regrammed Intermediate Cells KMEG-prCs

Previous study,a kind of reprogrammed intermediate cells,KMEG-prCs was achieved by c-Myc,Klf4,E-cadherin and Glis1 transduction,This cells are similar to embryonic stem cells in morphology,in vitro proliferation and differentiation in vitro and in vivo,but does not have chimeric ability.Although the cells have partial pluripotency,but oct4,sox2,nanog,AKP are not expressed,and only part cells were SSEA1 positive.By transcriptome analysis,KMEG-prCs are in reprogramming intermediate states.KMEG-prCs can be used as a new cellular model to study the molecular mechanism of the activation of endogenous pluripotent networks during reprogramming.This study was designed to utilize KMEG-prCs cells to screen for small molecule compounds or combinations that can activated the expression of endogenous pluripotent genes,and to lay the theoretical and technical foundation for further exploring the activation of endogenous pluripotent regulation network and the discovery of new small molecule compound combination to establish new chemical reprogramming system.Flow cytometry analysis showed that the surface marker proteins that the Thyl(CD90)was full down-regulated,CD54(ICAM-1)was full up-regulated,CD44 and SSEA1 were segmental up-regulation,and CD31 was only slightly up-regulated in KMEG-prCs.Based on the analysis of the transcriptome group,it was further determined that KMEG-prCs were intermediate cells at middle stage of reprogramming.SSEAl+KMEG-prCs and SSEA1-KMEG-prCs were sorted and cultured with the medium that containing different small molecule compounds.Endogenous pluripotent gene expression showed that the addition of HD AC inhibitor TSA,VPA,oct4 activator(OAC 1)and adenylate cyclase activator(Forskolin)activated the expression of pluripotency gene AKP,and a portion of SSEA1-cells were transformed into SSEAA1+ cells.Furtherly,the addition of EZH2 protein intracellular enhancer inhibitor(DZNeP)to induction,small molecule combination PCA+FD treatment can activate endogenous in SSEAl+KMEG-prCs and SSEA1-KMEG-prCs,and expressed oct4 at high level in SSEA1+KMEG-prCs,and obtained a new cell state,KMEG-prCs-O+,which still retains differentiation ability in vitro and in vivo.Subsequently,although A-83-01 in PCA+FD was replaced with E-616452,and combined with LiCl,PCE+FD+Li small molecule compound combination can effectively activate nanog,but oct4 was down-regulated.In summary,KMEG-prCs are intermediate cells that are in the middle of reprogramming,and their endogenous pluripotency regulation network can be activated by the treatment of small molecule compounds.This research provides a basis for the study of the mechanism of small molecule compounds for reprogramming and further re-programming of iPS cells,and also provides a basis for the study of cell signaling,metabolic pathways and epigenetic modifications during reprogramming.