Continuation of mammalian species requires the formation and development of the sexually dimorphic germ cells. Cultured embryonic stem cells are generally considered pluripotent rather than totipotent because of the failure to detect germline cells under differentiating conditions. Here we show that mouse embryonic stem cells in culture can develop into oogonia that enter meiosis, recruit adjacent cells to form follicle-like structures, and later develop into blastocysts. Oogenesis in culture should contribute to various areas, including nuclear transfer and manipulation of the germ line, and advance studies on fertility treatment and germ and somatic cell interaction and differentiation.

The four transcription factors Oct4, Sox2, Klf4, and c-Myc can induce pluripotency in mouse and human fibroblasts. We previously described direct reprogramming of adult mouse neural stem cells (NSCs) by Oct4 and either Klf4 or c-Myc. NSCs endogenously express Sox2, c-Myc, and Klf4 as well as several intermediate reprogramming markers. Here we report that exogenous expression of the germline-specific transcription factor Oct4 is sufficient to generate pluripotent stem cells from adult mouse NSCs. These one-factor induced pluripotent stem cells (1F iPS) are similar to embryonic stem cells in vitro and in vivo. Not only can these cells can be efficiently differentiated into NSCs, cardiomyocytes, and germ cells in vitro, but they are also capable of teratoma formation and germline transmission in vivo. Our results demonstrate that Oct4 is required and sufficient to directly reprogram NSCs to pluripotency.

In somatic cells, imprinted genes are expressed monoallelically according to parent-of-origin. In contrast, in 11.5 days post-coitum primordial germ cells (PGCs), and later stage germ cells, these same genes are expressed biallelically, suggesting that imprints inherited from the gametes are largely erased by this stage. To determine when in germ cell development this biallelic expression phenomenon commences, we isolated migrating PGCs by flow cytometry and determined the allele-specific expression of four imprinted genes - Snrpn, Igf2, H19 and Igf2r. The first three genes were expressed monoallelically, while the latter gene was expressed biallelically. These results show that inherited imprints regulating monoallelic expression are largely intact in migrating PGCs.

Primordial germ cells (PGCs) develop only into sperm and oocytes in vivo. The molecular mechanisms underlying human PGC specification are poorly understood due to inaccessibility of cell materials and lack of in vitro models for tracking the earliest stages of germ cell development. Here, we describe a defined and stepwise differentiation system for inducing pre-migratory PGC-like cells (PGCLCs) from human pluripotent stem cells (PSCs). In response to cytokines, PSCs differentiate first into a heterogeneous mesoderm-like cell population and then into PGCLCs, which exhibit minimal PRDM14 expression. PGC specification in humans is similar to the murine process, with the sequential activation of mesodermal and PGC genes, and the suppression of neural induction and of de novo DNA methylation, suggesting that human PGC formation is induced via epigenesis, the process of germ cell specification via inductive signals from surrounding somatic cells. This study demonstrates that PGC commitment in humans shares key features with that of the mouse, but also highlights key differences, including transcriptional regulation during the early stage of human PGC development (3–6 weeks). A more comprehensive understanding of human germ cell development may lead to methodology for successfully generating PSC-derived gametes for reproductive medicine.

The Oct-4 gene encodes a transcription factor that is specifically expressed in embryonic stem cells and germ cells of the mouse embryo. Cells that differentiate into somatic tissues lose Oct-4 expression. Regulation of Oct-4 gene transcription involves a TATA-less minimal promoter and two upstream elements: the proximal (PE) and distal enhancers (DE). We report here the nucleotide sequence of the 5' upstream regulatory regions of the human and murine Oct-4 genes. A comparative alignment analysis between these regions and those of the bovine Oct-4 ortholog reveals four conserved regions of homology (CR 1 to 4) between these species (66-94% conservation). The 1A sequence within the mouse PE is located approximately half-way between CR 2 and CR 3. A putative Sp1/Sp3 binding site and the overlapping hormone responsive element (HRE) in CR1 are identical in all three species. A high number of CCC(A/T)CCC motifs exhibit various levels of homology in these upstream regions. We discuss the importance of these and other sequences and present candidate factors that may bind and regulate Oct-4 gene expression.

SUMMARYOct1 (Pou2f1) is a transcription factor of the POU-homeodomain family that is unique in being ubiquitously expressed in both embryonic and adult mouse tissues. Although its expression profile suggests a crucial role in multiple regions of the developing organism, the only essential function demonstrated so far has been the regulation of cellular response to oxidative and metabolic stress. Here, we describe a loss-of-function mouse model for Oct1 that causes early embryonic lethality, with Oct1-null embryos failing to develop beyond the early streak stage. Molecular and morphological analyses of Oct1 mutant embryos revealed a failure in the establishment of a normal maternal-embryonic interface due to reduced extra-embryonic ectoderm formation and lack of the ectoplacental cone. Oct1 -/-blastocysts display proper segregation of trophectoderm and inner cell mass lineages. However, Oct1 loss is not compatible with trophoblast stem cell derivation. Importantly, the early gastrulation defect caused by Oct1 disruption can be rescued in a tetraploid complementation assay. Oct1 is therefore primarily required for the maintenance and differentiation of the trophoblast stem cell compartment during early post-implantation development. We present evidence that Cdx2, which is expressed at high levels in trophoblast stem cells, is a direct transcriptional target of Oct1. Our data also suggest that Oct1 is required in the embryo proper from late gastrulation stages onwards.

Patients with cystic fibrosis (CF) have decreased concentrations of expired nitric oxide (FENO) as compared with healthy individuals. A number of factors, including viscous mucus as a diffusion barrier for airway NO, consumption of NO by bacterial enzymes, and decreased NO production have been hypothesized to account for these low levels of FENO. We examined the relationship between the size of an AAT repeat polymorphism in intron 20 of the NOS1 gene and FENO in 75 patients with CF. Mean FENO was significantly (p = 0.027) lower in CF patients who harbored two alleles with a high number of repeats (>/= 12) than in those who harbored alleles with fewer repeats at this locus (4.0 +/- 0.8 [mean +/- SEM] ppb versus 6.4 +/- 0.9 ppb). Colonization of the airways with Pseudomonas aeruginosa was significantly (p = 0.0358) more common in CF patients with high numbers of AAT repeats in the NOS1 gene. Significant differences between NOS1 genotypes were also observed among patients homozygous for the cystic fibrosis transmembrane regulator delta F508 mutation for FENO (2.3 +/- 0.4 ppb versus 5.3 +/- 0.7 ppb, p = 0.0006), and this was also true for colonization of the airways with P. aeruginosa (p = 0.0147) and Aspergillus fumigatus (p = 0.0221). These data provide evidence that the NOS1 gene is not only associated with the variability of FENO, but also with P. aeruginosa colonization of airways in CF patients.

Molecular mechanisms underlying the commitment of cells to the germ cell lineage during mammalian embryogenesis remain poorly understood due to the limited availability of cellular materials to conduct in vitro analyses. Although primordial germ cells (PGCs)--precursors to germ cells--have been generated from embryonic stem cells (ESCs)--pluripotent stem cells derived from the inner cell mass of the blastocyst of the early embryo in vitro-the simultaneous expression of cell surface receptors and transcription factors complicates the detection of PGCs. To date, only a few genes that mark the onset of germ cell commitment in the epiblast--the outer layer of cells of the embryo--including tissue non-specific alkaline phosphatase (TNAP), Blimp1, Stella and Fragilis--have been used with some success to detect PGC formation in in vitro model systems. Here, we identified 11 genes (three of which are novel) that are specifically expressed in male and female fetal germ cells, both in vivo and in vitro, but are not expressed in ESCs. Expression of these genes allows us to distinguish committed germ cells from undifferentiated pluripotent cell populations, a prerequisite for the successful derivation of germ cells and gametes in vitro.