The recent development of porcine induced pluripotent stem cells (piPSCs) capable of generating chimeric animals, a feat not previously accomplished with embryonic stem cells or iPSCs in a species outside of rodents, has opened the doors for in-depth study of iPSC tumorigenicity, autologous transplantation, and other key aspects to safely move iPSC therapies to the clinic. The study of iPSC tumorigenicity is critical as previous research in the mouse showed that iPSCderived chimeras possessed large numbers of tumors, rising significant concerns about the safety of iPSC therapies. Additionally, piPSCs capable of generating germline chimeras could revolutionize the transgenic animal field by enabling complex genetic manipulations (e.g., knockout or knockin of genes) to produce biomedically important large animal models or improve livestock production. In this study, we demonstrate for the first time in a nonrodent species germline transmission of iPSCs with the live birth of a transgenic piglet that possessed genome integration of the human POU5F1 and NANOG genes. In addition, gross and histological examination of necropsied porcine chimeras at 2, 7, and 9 months showed that these animals lacked tumor formation and demonstrated normal development. Tissue samples positive for human POU5F1 DNA showed no C-MYC gene expression, further implicating C-MYC as a cause of tumorigenicity. The development of germline-competent porcine iPSCs that do not produce tumors in young chimeric animals presents an attractive and powerful translational model to study the efficacy and safety of stem cell therapies and perhaps to efficiently produce complex transgenic animals. STEM CELLS

Avian species are important model animals for developmental biology and disease research. However, unlike in mice, where clonal lines of pluripotent stem cells have enabled researchers to study mammalian gene function, clonal and highly proliferative pluripotent avian cell lines have been an elusive goal. Here we demonstrate the generation of avian induced pluripotent stem cells (iPSCs), the first nonmammalian iPSCs, which were clonally isolated and propagated, important attributes not attained in embryo-sourced avian cells. This was accomplished using human pluripotency genes rather than avian genes, indicating that the process in which mammalian and nonmammalian cells are reprogrammed is a conserved process. Quail iPSCs (qiPSCs) were capable of forming all 3 germ layers in vitro and were directly differentiated in culture into astrocytes, oligodendrocytes, and neurons. Ultimately, qiPSCs were capable of generating live chimeric birds and incorporated into tissues from all 3 germ layers, extraembryonic tissues, and potentially the germline. These chimera competent qiPSCs and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that were only previously available in mammals.

Early germ-like cells (GLCs) derived from human embryonic stem cells (hESCs) have presented new opportunities to study germ cell differentiation in vitro. However, differentiation conditions that facilitate the formation of haploid cells from the derived GLCs have eluded the field. The inability to propagate GLCs in culture is a further limitation, resulting in inconsistent rederivations of GLCs from hESCs with relatively few GLCs in these heterogeneous populations. Here we found in vitro conditions that enrich for DDX4/POU5F1+ GLCs (∼60%) and that has enabled continual propagation for >50 passages without loss of phenotype. Clonal isolation of single GLCs from these mixed cultures generated 3 GLC (>90% DDX4/POU5F1+) and 2 hESC (<0.1% DDX4+) lines that could be continually expanded without loss of phenotype. Differentiation of clonal GLC lines in serum resulted in expression of postmeiotic markers and >11% were haploid, ∼5-fold higher than previous studies. The robust clonal meiotic competent and incompetent GLC lines will be used to understand the factors controlling human germ cell meiosis and postmeiotic maturation.