Establishment And Optimization Of Differentiation Patterns Of Primate Embryonic Stem Cells Into Primordial Germ Stem Cells In Vitro

At present,more and more attention is paid to the research of animal reproductive development,and the understanding of germ cells has gradually deepened.However,the basic research on gamete formation,especially the study of primate gametes,is still insufficien.In vitro,culture system of pluripotent stem cells can help to accurately understand the mechanism of germ cell differentiation and functional germ cell development.The establishment of a non-human primate’s primordial germ stem cells differentiation model has established a viable research platform for humans,especially in the basic research of germ cells and the treatment of fertility diseases which is significance.The in vivo primordial germline stem cells are called PGCs,and the in vitro differentiated cells are called primary germ cell like cells(PGCLCs).In this dissertation,Primed ESCs were first differentiated into Na?ve ESCs,and next the Na?ve ESC induced Primordial germ cell like cell(PGCLC)in vitro.Optimized the differentiation pattern,analyzed the expression of omnipotent genes and primordial germline stem cell related genes at each differentiation stage to verified and proven monkey’s PGCLC characteristics.The results of the study are as follows.(1)The monkey Primed ESCs were first differentiated into Na?ve ESCs,which differentiation is stronger,using chemically transformed culture systems(including CHIR99021,PDO325901,SB203580,SP600125).Na?ve ESCs showed a rounded shape,expressed pluripotency transcription factors OCT4,SOX2,and NANOG.These cells also expressed the TRA-1-81 gene,what is a primate-specific pluripotent stem cell surface marker.It was confirmed that Primed ESCs were successfully differentiated into Na?ve ESCs.(2)A variety of cytokines were added to regulate cell differentiation(including BMP4,BMP2,LIF,SCF,EGF),and monkey Na?ve ESCs were used to differentiate PGCLCs through formation of embryoid bodies.It was determined that the optimal number of cells required for culturing embryoid bodies was 3000 cells per well,which used the 96-well U-type low adsorption cell culture plate.PGCLCs express pluripotent and conserved PGCs marker genes such as Blimp1,TFAP2 C,KIT and OCT4.It also expresses human-specific markers SOX15 and SOX17.Simultaneously,the relative expression of these specific genes was analyzed by qPCR,which also confirmed that the expression trends of multiple genes in monkey PGCLCs are consistent with the expression of PGCs in vivo.These results confirm that monkey Na?ve ESCs were successfully differentiated into PGCLCs.In summary,this thesis establishes and optimizes the differentiation pattern of monkey embryonic stem cells to primordial germline stem cells.It establishes and optimizes the differentiation pattern of Na?ve ESCs to PGCs,and proves that PGCLC expression trends of multiple genes are consistent with the characteristic gene expression profiles of PGCs in vivo.The results laid the foundation for the study of the formation and differentiation mechanism of primordial germ stem cells,and also laid the foundation for its application research and provided important data for the science of reproduction and development.