The germ-cell lineage ensures the continuity of life through the generation of male and female gametes, which unite to form a totipotent zygote.Wehave previously demonstrated that, by using cytokines, embryonic stem cells and induced pluripotent stem cells can be induced into epiblast-like cells (EpiLCs) and then into primordial germcell (PGC)-like cells with the capacity for both spermatogenesis and oogenesis1,2, creating an opportunity for understanding and regulatingmammalian germ-cell development in both sexesin vitro. Here we show that, without cytokines, simultaneous overexpression of three transcription factors, Blimp1 (also known as Prdm1), Prdm14 and Tfap2c (also known as AP2c), directs EpiLCs, but not embryonic stem cells, swiftly and efficiently into a PGC state. Notably, Prdm14 alone, but not Blimp1 or Tfap2c, suffices for the inductionof the PGC state inEpiLCs.The transcription-factor-induced PGC state, irrespective of the transcription factors used, econstitutes key transcriptome and epigenetic reprogramming in PGCs, but bypasses a mesodermal program that accompanies PGC or PGC-like-cell specification by cytokines including bone morphogenetic protein 4. Notably, the transcription-factor-induced PGC-like cells contribute to spermatogenesis and fertile offspring. Our findings provide a new insight into the transcriptional logic for PGC specification, and create a foundation for the transcription-factorbased reconstitution and regulation of mammalian gametogenesis.

The inductionkinetics by the TFs of endogenous Blimp1, Prdm14 and Tfap2c in whole EpiLC aggregates showed that (1) BLIMP1 andPRDM14activate Blimp1 gradually and Tfap2c rapidly, whereas TFAP2C does not activate Blimp1 nor Tfap2c, but enhances the activity of BLIMP1 to induce Tfap2c and Blimp1; (2) activation of Prdm14 by the TFs occurs relatively late (after 24 h); and (3) BP14A has the strongest effect on the activation of Blimp1, Tfap2c and subsequently Prdm14.

Notably, ten weeks after transplantation, the testes transplanted with the TF-PGCLCs, particularly those sorted at days 3 and 4, irrespective of the TFs used, contained numerous tubules with signs of spermatogenesis. These tubules showed normal spermatogenesis and contained spermatozoa with proper morphology. By contrast, SC ES cells induced by BP14A did not contribute to spermatogenesis, but formed foci of teratomas in six out of eight transplanted testes.