Derivation Of Male Germ Cell-like Cells From Porcine Induced Pluripotent Stem Cells

Differentiation and development of germ cells is a complicated and regulated process.The methods of induction of germ cells in vitro have important implications for treating infertility,conserving excellent animal breeds and studying germ cell development mechanisms.The study of induction of male germ cells from mouse and human pluripotent cells is more mature.However,the mouse has some deficiencies such as the short lifespan,and thus functional experiments of human germ cells cannot be long-time observed in mice.The functional experiments cannot be directly carried out in humans due to the ethical issues.Therefore,it is necessary to build the big animal model(such as pig)and then provide technology and safety evaluation systems for medicine.Compared with mouse,pig has a longer lifespan,as well as,pig has more similarities in physiology and anatomy with human.Moreover,pig does not have strict ethical issues and limits.Pig is thus an ideal animal model for medicine.In addition,pig is one of the most important livestock in the animal husbandry.So far,the research about porcine pluripotent cells needs further improvement.There are few reports about induction of germ cells from porcine pluripotent cells.Our research is focused on generating porcine induced pluripotent stem cells(iPSCs),and establishing methods of induction of male germ cell-like cells from porcine iPSCs.First of all,porcine iPSCs were generated.The porcine iPSCs were compact and dome-like colonies,which were morphologically similar to mouse embryonic stem cells.The porcine iPSCs were alkaline phosphatase-positive,and expressed pluripotency marker proteins including OCT4,SOX2 and SSEA-1.In vitro,the porcine iPSCs could form embryoid bodies and randomly differentiate into endoderm,mesoderm and ectoderm.Besides,the porcine iPSCs could differentiate into adipocyte-like cells and neuron-like cells.In immunodeficient mice,the porcine iPSCs can form teratoma that differentiated into three germ layers.The data demonstrate that porcine iPSCs have the potential of differentiation into germ cells.Next,we improved and optimized the methods of induction of mouse and human primordial germ cell-like cells(PGCLCs)according to porcine iPSCs characteristics,and then established the porcine germ cell-like cells induction methods.The porcine iPSCs were first transformed to epiblast stem cell-like cells(EpiLCs)in the medium containing cytokines such as Activin A.These EpiLCs highly expressed epiblast stem cell marker genes.Second,EpiLCs were differentiated into PGCLCs in the culture conditions including cytokines such as BMP4.PGCLCs significantly expressed primordial germ cells(PGCs)marker genes such as Stella,as well as,expressed PGCs marker proteins such as STELLA.In the epigenetic status,H3K27me3 levels were elevated,H3K9me2 levels were reduced,and imprinting genes became demethylated in the PGCLCs.These were similar to in vivo PGCs development.Transcriptome analysis showed that PGCLCs highly expressed genes associated with germ cell development.Third,PGCLCs were differentiated into spermatogonial stem cell-like cells(SSCLCs)in the induction culture system containing testosterone.SSCLCs expressed meiosis marker genes such as Stra8 and Gsg2,as well as,expressed spermatogonial stem cell marker proteins such as DAZL and VASA.Interestingly,haploid cells were detected in the SSCLCs population by flow cytometry.These results demonstrate that our induction systems could induce porcine iPSCs into male germ cell-like cells.There are no infertile pig models caused by the spermatogenic cells injury.In contrast,the methods of establishing infertile mice caused by the spermatogenic cells injury and the technology of seminiferous tubule injection are relatively mature.Mice were thus as recipient animals during in vivo exploration.PGCLCs and SSCLCs were transplanted into the seminiferous tubules of immunodeficient mice that lacked endogenous germ cells,and examined at variable time after transplantation.The results showed that induced germ cell-like cells formed chains and clusters in the mouse seminiferous tubules.Immunofluorescent staining data indicated germ cell marker proteins including DAZL and VASA were positive.These data demonstrate that cells after transplantation exhibited germ cell features and may contribute to repairing the testis with the spermatogenic cells injury.Taken together,porcine iPSCs that were similar to mouse embryonic stem cells were derived in our research.Furthermore,our study provided a feasible strategy for directing porcine iPSCs to male germ cell-like cells.The study lays the foundation for exploring germ cell development mechanisms and differentiating human pluripotent cells into germ cells clinically,as well as,provides new ideas for animal breeding in the animal husbandry.