Derivation of pluripotent stem cells from nascent undifferentiated teratoma.

Teratomas are tumors consisting of components of the three germ layers that differentiate from pluripotent stem cells derived from germ cells. In the normal mouse testis, teratomas rarely form, but a deficiency in Dead-end1 (Dnd1) in mice with a 129/Sv genetic background greatly enhances teratoma formation. Thus, DND1 is crucial for suppression of teratoma development from germ cells. In the Dnd1 mutant testis, nascent teratoma cells emerge at E15.5. To understand the nature of early teratoma cells, we established cell lines in the presence of serum and leukemia inhibitory factor (LIF) from teratoma-forming cells in neonatal Dnd1 mutant testis. These cells, which we designated cultured Dnd1 mutant germ cells (CDGCs), were morphologically similar to embryonic stem cells (ESCs) and could be maintained in the naïve pluripotent condition. In addition, the cells expressed pluripotency genes including Oct4, Nanog, and Sox2; differentiated into cells of the three germ layers in culture; and contributed to chimeric mice. The expression levels of pluripotency genes and global transcriptomes in CDGCs as well as these cells' adaption to culture conditions for primed pluripotency suggested that their pluripotent status is intermediate between naïve and primed pluripotency. In addition, the teratoma-forming cells in the neonatal testis from which CDGCs were derived also showed gene expression profiles intermediate between naïve and primed pluripotency. The results suggested that germ cells in embryonic testes of Dnd1 mutants acquire the intermediate pluripotent status during the course of conversion into teratoma cells.

SETDB1 is essential for mouse primordial germ cell fate determination by ensuring BMP signaling.

In mouse embryos, primordial germ cells (PGCs) are fate-determined from epiblast cells. Signaling pathways involved in PGC formation have been identified, but their epigenetic mechanisms remain poorly understood. Here, we show that the histone methyltransferase SETDB1 is an epigenetic regulator of PGC fate determination. Setdb1-deficient embryos exhibit drastic reduction of nascent PGCs. Dppa2, Otx2 and Utf1 are de-repressed whereas mesoderm development-related genes, including BMP4 signaling-related genes, are downregulated by Setdb1 knockdown during PGC-like cell (PGCLC) induction. In addition, binding of SETDB1 is observed at the flanking regions of Dppa2, Otx2 and Utf1 in cell aggregates containing PGCLCs, and trimethylation of lysine 9 of histone H3 is reduced by Setdb1 knockdown at those regions. Furthermore, DPPA2, OTX2 and UTF1 binding is increased in genes encoding BMP4 signaling-related proteins, including SMAD1. Finally, overexpression of Dppa2, Otx2 and Utf1 in cell aggregates containing PGCLCs results in the repression of BMP4 signaling-related genes and PGC determinant genes. We propose that the localization of SETDB1 to Dppa2, Otx2 and Utf1, and subsequent repression of their expression, are crucial for PGC determination by ensuring BMP4 signaling.

Repression of Somatic Genes by Selective Recruitment of HDAC3 by BLIMP1 Is Essential for Mouse Primordial Germ Cell Fate Determination.

Primordial germ cells (PGCs) are fate determined from pluripotent epiblasts. Signaling pathways and transcriptional regulators involved in PGC formation have been identified, but detailed molecular mechanisms of PGC fate determination remains poorly understood. Using RNAi screening, we identified histone deacetylase 3 (HDAC3) as a regulator of PGC formation. Hdac3 deficiency resulted in decreased nascent PGCs in vitro and in vivo, and somatic developmental genes were de-repressed by Hdac3 knockdown during PGC induction. We also demonstrated BLIMP1-dependent enrichment of HDAC3 and deacetylation of H3 and H4 histones in the somatic developmental genes in epiblast-like cells. In addition, the HDAC3/BLIMP1-targeted somatic gene products were enriched in PGC determinant genes; overexpression of these gene products in PGC-like cells in culture resulted in repression of PGC determinant genes. We propose that selective recruitment of HDAC3 to somatic genes by BLIMP1 and subsequent repression of these somatic genes are crucial for PGC fate determination.

Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment.

Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs.