show Abstracthide AbstractPluripotent stem cells can be obtained from spermatogonial stem cells (SSCs) through spontaneous reprogramming without transgenic introduction, but this process is inefficient and lacks mechanism exploration. Here, we reconstructed the progression trajectory of mouse SSC reprogramming by single-cell RNA sequencing and identified a bona fide pluripotent route as the successful reprogramming branch. Notably, we developed five-chemicals combination which could boost the reprogramming efficiency nearly 1000 times than previous study. More importantly, chemical induced germline-derived pluripotent stem cells (5C-gPSCs) with embryonic stem cell-like imprinting status were characterized as fully pluripotent via tetraploid complementation assay. Mechanistically, not only the expression of canonical markers, but also the global DNA demethylation and re-methylation features from epiblast to primordial germ cells and subsequent spermatogonia were remarkably observed during SSC reprogramming; meanwhile, biallelic methylation of most imprinting control regions (ICRs) in SSCs had been switched to monoallelic methylation in gPSCs through such stepwise reprogramming process. Compared to the extremely hypomethylated ICRs in developmental deficient chemical induced pluripotent stem cells (CiPSCs), we demonstrated the highly correlation between proper methylation of ICRs and fully pluripotency. Therefore, our work sheds light on the unique genetic- and epigenetic regulatory network of SSC reprogramming, providing novel insights to understand generic mechanisms for cell fate determination and the epigenetic related developmental disorders in regenerative medicine. Overall design: In this study, we established a highly efficient SSC reprogramming system to generate fully pluripotent gPSCs via chemical induction, moreover, with the aid of genetic- and epigenetic dissections using single-cell multi-omics (transcriptome and DNA methylome) sequencing, we explored the sophisticated regulation network of cell fate transition during this reprogramming process. SSCR: Spermatogonial stem cell reprogramming; gPSC: germline derived plueipotent stem cells; CiPS: chemical induced pluripotent stem cell.