Regulation Of Pre-iPSCs Reprogramming In G1 Phase

Background: Since the establishment of induced pluripotent stem cells(iPSCs),it is now possible to reprogram differentiated adult cells into pluripotent cells similar to embryonic stem cells.The technology promises prospect for the field of stem cells and regenerative medicine.However,the process of induced pluripotent stem cells is complex and the mechanism is not fully understood.In the process of somatic reprogramming,not all cells can be induced to become pluripotent stem cells.In fact,most cells have characteristic morphology similar to that of pluripotent stem cells,but their core pluripotent network is not activated and the core pluripotent gene is not expressed,which is called pre-iPSCs.In addition,cell proliferation and differentiation cannot be separated from cell cycle.More and more studies have shown that the proliferation rate and cell cycle of different types of cells are significantly different.All these discoveries suggest that iPSCs are closely related to the cell cycle,and the study of iPSCs and the cell cycle may reveal the unknown mysteries of the reprogramming mechanism.Objective: 1,To compare the proliferation rates of different pluripotent cells,we strive to explore the intercellular relationship between the cell cycle.2,Facilitate preiPSCs transform into iPSCs by regulating cell cycle relative genes.3,Establish a novel reprogramming system and explore the connection between rapid reprogramming and cell cycle.Exploring how pre-iPSCs can be induced into pluripotent stem cells may be a new way to explore the reprogramming mechanism.Method: the proliferation rates of different cell types were analyzed by cell count.The cell cycle distribution of each cell line was analyzed by PI staining and fluorescence activated cell sorting(FACS).RNA-seq of different systems was constructed,and the general association of cell cycle related genes was obtained through RNA-seq data analysis.Construct overexpression and knockdown plasmid vectors and regulate gene of cells by using lentivirus packaging system.RNA extraction and qPCR were used to verify the correct expression of related genes.A new reprogramming induction system was constructed to compare the relationship between rapid reprogramming and cell cycle.Result: In this study,we first compare the proliferation rates of different types of cells,including OG2 ES,R1 ES,and pre-iPSCs,etc.and it was found that the proliferation rate of pre-iPSCs was the fastest.In addition,PI staining was used to further explore the cell cycle in combination with FACS,and dates showed that preiPSCs experienced a longer G1 phase.In order to further analyze how cell cycle regulates pluripotency,this paper compared multiple databases and conducted corresponding qPCR verification for cell cycle genes regulating G1 phase.Finally,two groups of G1 phase regulation systems were designed,shCcnd1?shCcnd2?shCdk6?Cdk4 for G1 phase,and Ccne1?Ccne2?Cdk2 for G1/S checkpoint.After regulating the corresponding cell cycle genes of pre-iPSCs,green fluorescence cloning was produced about 12 days culture,and the pluripotent genes such as Sox2,Nanog,Rex1 and Dppa3 was activated verified by qPCR.In addition,the purpose of this study was to investigate whether faster reprogramming is related to the cell cycle.Therefore,a new reprogramming system OvSvK was constructed.Transcription activator VP16 was connected with Oct4 and Sox2.In this system,the level of induced reprogramming on day 4 can reach the same level as that on day 7 of OSK reprogramming.Besides,OvSvK reprogramming products more OCT4-GFP+ colonies than OSK reprogramming,proving that the OvSvK system can induce a more rapid reprogramming.Cell cycle analysis showed that the proportion of G1 phase in the reprogramming process was gradually reduced.And cell cycle data for other kind of cells also showed that cells with higher pluripotency experienced a shorter G1 phase.Conclusion: In a word,this study revealed that G1 phase of cell cycle is an obstacle to somatic reprogramming,and the regulation of G1 phase enable reactivate the pluripotency of pre-iPSCs and make them transform into iPSCs,indicating the importance of cell cycle for reprogramming,and providing a new idea for explore the mechanism of cell reprogramming.