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The ability to generate germ cells from pluripotent stem cells (PSCs) is valuable for human regenerative medicine and animal breeding. Germ cell-like cells (GCLCs) have been differentiated from mouse and human PSCs, but not from porcine PSCs, which are considered an ideal model for stem cell applications. Here, we developed a defined culture system for the induction of primordial germ cell-like cells (PGCLCs) from porcine induced PSCs (piPSCs). The identity of the PGCLCs was characterized by observing cell morphology, detecting germ cell marker gene expression and evaluating epigenetic properties. PGCLCs could further differentiate into spermatogonial stem cell-like cells (SSCLCs) in vitro. Importantly, meiosis occurred during SSCLC induction. Xenotransplantation of GCLCs into seminiferous tubules of infertile immunodeficient mice resulted in immunohistochemically identifiable germ cells in vivo. Overall, our study provides a feasible strategy for directing piPSCs to the germ cell fate and lays a foundation for exploring germ cell development mechanisms.

Induced pluripotent stem cells (iPSCs) have variable expression levels of a series of genes that affect their pluripotent potential, but the regulatory mechanisms controlling reprogramming remain unclear. By testing the efficiency of iPSC generation using Oct4, Sox2, Klf4 (termed OSK) plus one additional gene, we found that Rab32 improved reprogramming efficiency. We established a system for detecting the number and the size of lipid droplets and autophagosomes per cell for tracking their morphological changes during reprogramming. Our results showed that Rab32 increased lipid storage during the early and middle stages and also increased autophagy during the middle stage of reprogramming. These findings were further confirmed by the up-regulation of lipid biosynthesis and autophagosome formation related genes, of which their expression could improve iPSC induction. The inhibition of lipid biosynthesis and autophagosome formation significantly reduced reprogramming efficiency and the inhibition of lipid synthesis phenotype could be rescued by the overexpression of Rab32. In addition, the expression of pluripotency genes such as Klf2, Nr5a2 and Tbx3, was up-regulated by Rab32. These results demonstrated that Rab32 could improve the induction of iPSCs through the enhancement of lipid biosynthesis, highlighting the importance of lipid metabolism during reprogramming.