| Somatic cells can be reprogrammed to pluripotent stem cells,which provides thrapeutic root cells for regenerative medicine.Compared to NT-ESCs and iPSCs,chemically induced pluripotent stem cells(CiPSCs)avoided the use of oocytes or genetic manipulation,making cell fate transitions potentially easier to control in a costeffective and nonimmunogenic way.However,authentic pluripotency of CiPSCs has never been achieved through tetraploid complementation assay.Spermatogonial stem cells(SSCs)locate in the testis and initiate continuous spermatogenesis,these SSCs can be well maintained in vitro for several passages.Intriguingly,in culture,a tiny majority of SSCs spontaneously convert to germline-derived pluripotent stem cells(gPSCs),in a form of non-transgenic reprogramming,however,the underlying mechanism triggerring cell fate transition remained largely unknown.In previous study,we have mapped the cell fate trajectory of SSCs reprogramming using single-cell RNA sequencing,found that GO terms such as ’glycolysis’ and ’DNA methylation/demethylation’ were enriched in the successful branch.Based on the transcriptome analysis and previous studies,a combination of five chemicals(named 5C,SGC707,Vitamin C,EGCG,TUDCA and Daphnetin)were identified to improve the efficiency of SSC by nearly 100-folds and accelerated the reprogramming progress as early as day 10.Besides the efficiency improvement,we also wondered whether the chemical treatment was beneficial to the developmental potency of gPSCs.To this end we performed tetraploid complementation assay(’gPSC 4N-comp’).When Oct4-EGFP PSCs were injected into tetraploid ICR blastocysts,live pups could be developed from gPSCs and 5C-gPSCs.However,4N-comp mice derived from gPSCs showed heavier neonatal body weight,much lower survival rate and growth curve than that from 5CgPSCs and ESCs.DNA methylation analysis by bisulfite-sequencing demonstrated that the proper methylation level of whole genome and total imprinting control regions(ICRs)were observed in ESCs and 5C-gPSCs,but not in gPSCs.Besides,the DNA methylation of Dlk1-Dio3 region and miRNAs encoded locus in this cluster preserved a relatively moderate level in ESCs and 5C-gPSCs.Together,these data suggested a positive-correlation between proper imprinting status of 5C-gPSCs and their subsequent developmental and growth advantages in tetraploid-derived mice.To further decode the regulatory mechanism of SSCs reprogramming,single-cell multi-omics(transcriptome and DNA methylome)sequencing was performed.Mechanistically,we found that the reprogramming pathway from SSC to PSC resembled an inverted pathway of germ cell development in vivo,from the aspects of gene expression and DNA methylation dynamics from spermatogonia to primordial germ cells(PGCs)and further to Epiblasts.Besides,in the late stage of SSCs reprogramming,gPSCs re-established the global DNA methylation and methylation of most maternal ICRs(including Igf2r,Snrpn,Impact,Peg3,Peg10,and Plagl1),which were erasured in the early.Impressively,allele split further verified the re-methylation of maternal ICRs(Impact,Peg10,and Snrpn)of one of the two alleles in one cell during the late stage of reprogramming.What’s more,the epigenetic features(DNA methylation)were robust in 5C condition than the control.In conclusion,we confiremed that 5C-gPSCs are truly pluripotent stem cells determined by tetraploid compensation assay and 5C can help to repair the epigenetic and developmental defects.In addition,we showed that SSC reprogramming traversed through an inverted route of in vivo germ cell development and the chemical-induced gPSCs had correct imprinting status using single-cell multi-omics(transcriptome and DNA methylome)analysis.Finally,this unique trajectory and regulatory network of SSC reprogramming described here expands our understanding of cell fate reprogramming,such knowledge paves the way to obtain high-quality iPSCs without genetic manipulation for future clinical applications in regenerative medicine. |
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