Comparative Analysis Of Gene Regulation Differences Between Different Somatic Cell Reprogramming Technology In Mammalian Based On Single-cell RNA-seq

Somatic cell reprogramming technology has great potential in stem cell biology,disease modeling,therapeutic cloning,but the barriers affecting the somatic cells reprogramming efficiency and the differences between different somatic cell reprogramming technologies remain incompletely elucidated.In this study,we took the reprogramming process of somatic cell nuclear transfer(SCNT)and induced pluripotent stem cells(iPSC)in bovine and mouse as research objects,systematically exploring the molecular processes of cell fate transition during somatic cell reprogramming and gene regulatory differences between different somatic cell reprogramming systems by single-cell RNA sequencing and integrating related data in public database.First,we conducted sampling and single-cell RNA sequencing at each preimplantation development stage of in vitro fertilization(IVF)embryos and nuclear transfer embryos with somatic cells and iPSC nuclei as donors,respectively.The study results showed that bovine SCNT embryos were highly similar to IVF embryos in terms of single-cell clustering,dynamic waves of differentially expressed genes(DEGs),and marker genes of developmental stages.Nonetheless,many abnormal expression genes were found in each development stage of SCNT embryos,which mainly enriched in metabolic activity,epigenetic modification,organelle activity etc.Compared with IVF embryos,the expression pattern of DEGs in SCNT embryos lacked strict temporal regulation.And SCNT embryos exhibited a series of genes inhibition(including ZSCAN4)as well as defects in functional protein association networks at 8-cell stage.In addition,we also found that iPSC nuclear transfer(iPSCNT)embryos had more aberrantly expressed genes and exhibited a distinct pseudotime trajectory from IVF and SCNT embryos.KDM family and a gene cluster including DDX56,DNM2 down regulated expression along the specific pseudotime trajectory of iPSCNT embryos.This indicated that iPSC nuclei as donors hindered the reprogramming of nuclear transfer embryos.To further reveal gene regulation differences between different somatic cell reprogramming technologies,we integrated the single-cell transcriptome data of mouse SCNT and iPSC from the public databases.The cell clustering results indicated that preimplantation SCNT embryos can be divided into two stages,before and after major zygotic genome activation(ZGA),and the process of iPSC derivation from somatic cells can be divided into three stages:prophase,metaphase,and anaphase.After the major ZGA,SCNT embryos established a more complex gene regulatory network with Tbx20 and other transcription factors(TFs)as the core and activated a series of downstream functional pathways.Whereas during iPSC derivation,genes such as Kdm5b,Klf4,Nanog,Tdgf1 were activated as key factors of cell fate determination upon OSKM withdrawal and turned on the downstream pluripotency molecular networks to enable the cells to be properly reprogrammed into iPSCs.We found pluripotency and epigenetic modifiers such as Dppa2/4,Obox6,Sox2,Kdm5b,which played key roles in iPSCs,were also activated at different stages in SCNT embryos and coordinately regulated to promote the reprogramming of SCNT embryos.Whereas embryonic ZGA associated genes other than Dppa2/4 do not appear to be essential during iPSC reprogramming.In conclusion,we deeply revealed the abnormal gene expression in SCNT embryos and gene regulation differences between SCNT and iPSC reprogramming processes from single-cell clustering,comparison of genes differential expression patterns,marker genes identification,mechanisms of cell fate determination,gene co-expression analysis,TF regulatory network identification.Our study provided new insights into understanding different somatic cell reprogramming technology and improving somatic cells reprogramming efficiency.