Karin Hübner

OCT4-INDUCED PLURIPOTENCY IN ADULT NEURAL STEM CELLS

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.

Allele-specific expression of imprinted genes in mouse migratory primordial germ cells

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.

Comparative analysis of human, bovine, and murine Oct-4 upstream promoter sequences

Abstract. 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 CR 1 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.

Human primordial germ cell commitment in vitro associates with a unique PRDM14 expression profile

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.

Oct1 regulates trophoblast development during early mouse embryogenesis

Oct1 (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.

Identification of genes specific to mouse primordial germ cells through dynamic global gene expression

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.

Spermatogonia: origin, physiology and prospects for conservation and manipulation of the male germ line

In recent years, the scientific community has become increasingly interested in spermatogonia. Methodological breakthroughs, such as germ cell transplantation and spermatogonial culture combined with novel germ line transfection strategies, have provided interesting new opportunities for studying the physiology of spermatogonial stem cells and their interaction with the stem cell niche. Furthermore, intense research into pluripotent and adult stem cells has generated new insight into the differentiation pathway of germ line stem cells and has opened new perspectives for stem cell technologies. The present review briefly introduces the physiology of spermatogonial stem cells and discusses future directions of basic research and practical approaches applicable to livestock maintenance and animal reproduction.

Ultrastructural characterization of mouse embryonic stem cell-derived oocytes and granulosa cells.

Germ cells are a unique population of cells responsible for transmitting genetic information from one generation to the next. Our understanding of the key mechanisms underlying germ cell development in vivo remains scarce because of insufficient amounts of cell materials available for conducting biological studies. The establishment of in vitro differentiation models that support the generation of germ cells from mouse pluripotent stem cells provides an alternative means for studying reproductive development. The detection and analysis of stem cell-derived germ cells, however, present technical challenges. Methods for determining the developmental stage of germ cells ex vivo, such as gene expression and/or immunochemical analyses are inadequate, frequently necessitating the use of alternative, elaborate methods to prove germ cell identity. We have generated putative oocytes and granulosa cells in vitro from mouse embryonic stem cells and utilized electron microscopy to characterize these cells. Here, we report on the striking ultrastructural similarity of in vitro-generated oocytes and granulosa cells to in vivo oocytes developing within follicles.

Germ Cell Nuclear Factor Regulates Gametogenesis in Developing Gonads

Expression of germ cell nuclear factor (GCNF; Nr6a1), an orphan member of the nuclear receptor gene family of transcription factors, during gastrulation and neurulation is critical for normal embryogenesis in mice. Gcnf represses the expression of the POU-domain transcription factor Oct4 (Pou5f1) during mouse post-implantation development. Although Gcnf expression is not critical for the embryonic segregation of the germ cell lineage, we found that sexually dimorphic expression of Gcnf in germ cells correlates with the expression of pluripotency-associated genes, such as Oct4, Sox2, and Nanog, as well as the early meiotic marker gene Stra8. To elucidate the role of Gcnf during mouse germ cell differentiation, we generated an ex vivo Gcnf-knockdown model in combination with a regulated CreLox mutation of Gcnf. Lack of Gcnf impairs normal spermatogenesis and oogenesis in vivo, as well as the derivation of germ cells from embryonic stem cells (ESCs) in vitro. Inactivation of the Gcnf gene in vivo leads to loss of repression of Oct4 expression in both male and female gonads.

Derivation of germ cells from embryonic stem cells.

Embryonic stem cells (ESCs), derivatives of cells of early mammalian embryos, have proven to be one of the most powerful tools in developmental and stem cell biology. When injected into embryos, ESCs can contribute to tissues derived from all three germ layers and to the germline. Prior studies have successfully shown that ESCs can recapitulate features of embryonic development by spontaneously forming somatic lineages in culture. Amazingly, recently it has been shown that mouse ESCs can also give rise to primordial germ cells (PGCs) in culture that are capable of undergoing meiosis and forming both male and female gametes. While the full potential of these ES-derived germ cells and gametes remains to be demonstrated, these discoveries provide a new approach for studying reproductive biology and medicine.