The Application Of Three-dimensional Culture In Vitro Differentiation Of Mouse Primordial Germ Cells

Primordial germ cells are the earliest germline cells generated in the body,which have the potential to differentiate to sperm or oocyte.Mouse embryonic stem cells can differentiate into mouse primordial germ cells-like cells(m PGCLCs)through epiblast like cells stage in certain conditions in vitro.With the stimulation of retinoic acid,testosterone,gonadotropin pituitary extract and etc.,the m PGCLCs cultured with sertoli cells,which obtained from newborn c-kit mouse,can complete the meiosis and obtain the round spermatids.Although we can obtain the round spermatids through in vitro differentiation,the efficiency of m PGCLCs in vitro differentiation system is still low,which means that we still cannot solve the problem of large scale acquisition of target cells.In recent years,3-dimensional cell culture technique becomes the hotspot of cell culture research since this technique can not only simulate in vivo environment more accurately,but also obtain the target cells more efficiently.Significantly,the3-dimensional cell culture technique has not been reported in the research of mammalian germ cells differentiation in vitro.The method of this experiment is an optimization of traditional method.Since the viscous trait of methylcellulose(MC)can decrease the precipitation velocity of cell sphere,methylcellulose was added to generate a simple 3-dimentional cell culture environment in this experiment.Furthermore,the differentiation experiment process was optimized as well.These works established foundation of the 3-dimensional cell in vitro differentiation system.Our work performs the transgenic m ESCs with co-expression of BPT(Blimp1,Prdm14 and Tfap2c)to finish the differentiation in vitro.0.35% MC was added in the culture medium to proceed the 3-dimensional culture in Epi LCs and m PGCLCs induction stage.Immunofluorescence staining of the Epi LCs stage shows that the expression of Oct4 match the characteristics of this stage,after comparing with the traditional culture method,flow cytometry and fluorescence sorting technology(Fluoresvemce activated Cell Sorting,FACS)analysis showed no differentiation efficiency of modified m PGCLCs induction method.Further real-time PCR analysis of the key gene expression profiles of day4 m PGCLCs,the expression levels of the pluripotent genes and the PGCs gene were consistent with the results of the traditional culture.The results showed that the m PGCLCs state obtained from the optimized experiment was normal,but the differentiation efficiency was not improved.However,it was found that the yield of m PGCLCs was increased by about 3 times under the same amount of medium.Although the project did not do further functional experiments,a large number of differentiated cells in vitro can be obtained through this method,which provides the initial methods and technical support.