Study On Biological Characteristics And Differentiation In Vitro Of Mule Induced Pluripotent Stem Cells

The mule is the hybrid born from the horse and the donkey,and most of them are infertility.The hypotheses of mule fertility regulation have been put forward,but there is no definite evidence.In 2011,Saitou et al.induced mouse embryonic stem cells(ESCs)into primordial germ cell-like cells(PGCLCs)and successfully obtained surviving offspring,and also similar to mouse iPSCs.In subsequent studies,different methods can be used to induce human and equine iPSCs into PGCLCs.This study tried to use the advantages of fertile mule iPSCs established in our laboratory into PGCLCs,in order to provide a scientific basis for studying the reproductive biological mechanism of mule infertility.The results are shown below:1.FMF-iPSCs had a large ratio of nucleus to cytoplasm,and the cells were closely connected,the clone morphology was irregular and flat,with clear boundaries and positive alkaline phosphatase staining.2.The results of karyotype analysis showed that the number of chromosomes of FMF-iPSCs was normal and the genome was stable,2N=63/XX.3.The results of real-time fluorescent quantitative PCR and immunofluore scence staining showed that FMF-iPSCs expressed pluripotency marker genes OCT4,SOX2,NANOG in RNA and protein levels.4.EBs differentiation in vitro showed that FMF-iPSCs have the potential to differentiate into endoderm,mesoderm and ectoderm in vitro,HNF4 A,HAND1 and NESTIN were expressed at the RNA level,and followed analysis GATA6,T and NESTIN were expressed at the protein level,respectively.5.FMF-iPSCs were injected into the mouse Epiblast embryos of 6.5 days,which proliferated and migrated after 48 hours cultured in vitro,showing the potential to contribute to endoderm,mesoderm,ectoderm and PGCs.6.PGCLCs directed differentiation results showed that,use three methods,FMF-iPSCs can be induced into PGCLCs,PGCs genes marker NANOS3,BLIMP1,SOX17 were expressed at the RNA level,and followed analysis VASA,BLIMP1 were expressed at the protein level,respectively.In summary: this study has successfully obtained fertile female mule iPSCs that have been passaged stably in the serum Li F culture system for a long time,and it has self-renew ability and maintains stable genomic characteristics.This mule iPSCs have the potential to differentiate into endoderm,mesoderm,ectoderm and PGCs in vitro,following injected it into the mouse Epiblast embryos for 6.5 days and transgenetic treatment.This study successfully established the mule iPSCs and identified the ability of tridermogenesis and PGCs differentiation,which is important for cross breeding and animal evolution in the future research.