RNA interference (RNAi) is a sequence-specific gene regulatory mechanism conserved among diverse eukaryotes. The sequence specificity in RNAi is determined by a family of 18-to 30-nucleotide (nt) regulatory small RNAs (for review, see Aravin and Tuschl 2005). Two major classes of endogenous small RNAs have been characterized: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs-the best-characterized endogenous small RNAs in eukaryotes-have been identified in diverse plants and animals, and are mainly involved in development and differentiation. miRNAs are processed from miRNA precursors (pre-miRNAs) with a stem-loop structure and regulate gene expression through translational repression or mRNA cleavage (for reviews, see Ambros 2004;Bartel 2004;He and Hannon 2004;Du and Zamore 2005). siRNAs are generated from long double-stranded RNA (dsRNA) and are mainly involved in defense against molecular parasites including viruses, transposons, and transgenes through RNAi (Sijen and Plasterk 2003;Shi et al. 2004). Endogenous siRNAs have been classified into at least three subclasses: repeat-associated siRNAs (rasiRNAs), trans-acting siRNAs (ta-siRNAs), and siRNAs derived from natural antisense transcripts (nat-siRNAs) (Lippman and Martienssen 2004;Peragine et al. 2004;Borsani et al. 2005). rasiRNAs corresponding to repetitive elements repress the repeat sequences at the transcriptional or post-transcriptional level and maintain a centromeric heterochromatic

The inner cell mass (ICM) and trophoblast cell lineages duet early embryonic development in mammals. After implantation, the ICM forms the embryo proper as well as some extraembryonic tissues, whereas the trophoectoderm (TE) exclusively forms the fetal portion of the placenta and the trophoblast giant cells. Although embryonic stem (ES) cells can be derived from ICM in cultures of mouse blastocysts in the presence of LIF and/or combinations of small-molecule chemical compounds, and the undifferentiated pluripotent state can be stably maintained without use of serum and feeder cells, defined culture conditions for derivation and maintenance of undifferentiated trophoblast stem (TS) cells have not been established. Here, we report that addition of FGF2, activin A, XAV939, and Y27632 are necessary and sufficient for derivation of TS cells from both of E3.5 blastocysts and E6.5 early postimplantation extraembryonic ectoderm. Moreover, the undifferentiated TS cell state can be stably maintained in chemically defined culture conditions. Cells derived in this manner expressed TS cell marker genes, including Eomes, Elf5, Cdx2, Klf5, Cdh1, Esrrb, Sox2, and Tcfap2c; differentiated into all trophoblast subtypes (trophoblast giant cells, spongiotrophoblast, and labyrinthine trophoblast) in vitro; and exclusively contributed to trophoblast lineages in chimeric animals. This delineation of minimal requirements for derivation and self-renewal provides a defined platform for precise description and dissection of the molecular state of TS cells.

Nuclear transfer has been regarded as the only reliable tool for studying nuclear reprogramming of mammalian somatic cells by oocytes. However, nuclear transfer is not well suited for biochemical analyses of the molecular mechanisms of reprogramming. A cell-free system from oocytes is an attractive alternative way to mimic reprogramming in vitro, since a large number of cells can be treated and analyzed. Nevertheless, a cell-free system using oocytes has not been developed in mammals. Here, cell extracts from porcine oocytes were prepared and their ability to induce nuclear reprogramming was evaluated. Extracts from metaphase II (MII) oocytes erased the machinery for regulating gene expression in reversibly permeabilized somatic cells. For example, the extracts caused histone deacetylation and the disappearance of TATA box-binding protein from the nuclei. However, MII-extract-treated cells did not show any obvious changes after cell culture. In contrast, extracts from germinal vesicle (GV) oocytes activated pluripotent marker genes, especially NANOG, and induced partial dedifferentiation after cell culture. The activation of pluripotent marker genes by GV extracts was associated with histone acetylation that was induced during extract treatment. These results indicate that GV- and MII-oocyte extracts have different roles on nuclear reprogramming. Furthermore, both oocyte extracts induced site-specific demethylation in the upstream region of NANOG. These results indicate that cell-free extracts derived from GV- and MII-oocytes could be useful for studying the mechanisms involved in nuclear reprogramming.

Generation of pluripotent stem cells (PSCs) in large domestic animals has achieved only limited success; most of the PSCs obtained to date have been classified as primed PSCs, which possess very little capacity to produce chimeric offspring. By contrast, mouse PSCs have been classified as naïve PSCs that can contribute to most of the tissues of chimeras, including germ cells. Here, we describe the generation of two different types of bovine induced pluripotent stem cells (biPSCs) from amnion cells, achieved through introduction of piggyBac vectors containing doxycycline-inducible transcription factors (Oct3/4, Sox2, Klf4, and c-Myc). One type of biPSCs, cultured in medium supplemented with knockout serum replacement (KSR), FGF2, and bovine leukemia inhibitory factor (bLIF), had a flattened morphology like human PSCs; these were classified as primed-type. The other type biPSCs, cultured in KSR, bLIF, Mek/Erk inhibitor, GSK3 inhibitor and forskolin, had a compact morphology like mouse PSCs; these were classified as naïve-type. Cells could easily be switched between these two types of biPSCs by changing the culture conditions. Both types of biPSCs had strong alkaline phosphatase activity, expressed pluripotent markers (OCT3/4, NANOG, REX1, ESRRβ, STELLA, and SOCS3), and formed embryoid bodies that gave rise to differentiated cells from all three embryonic germ layers. However, only naïve-type biPSCs showed the hallmarks of naïve mouse PSCs, such as LIF-dependent proliferation, lack of FGF5 expression, and active XIST expression with two active X chromosomes. Furthermore, naïve-type biPSCs could contribute to the inner cell mass (ICM) of host blastocysts and most tissues within chimeric embryos. This is the first report of generation of biPSCs with several characteristics similar to those of naïve mouse PSCs and a demonstrated potential to contribute to chimeras.

Plasma membranes can be reversibly permeabilized by Streptolysin O. The permeabilized cells can be reprogrammed and partially dedifferentiated in the cell-free system from egg extracts. However, the permeabilizing activity of Streptolysin O is not stable, and therefore it is difficult to control its activity. An alternative method for reversible permeabilization is useful for establishing a cell-free system. Here, we used a nonionic detergent, digitonin, for permeabilization. A low concentration of digitonin induced reversible permeabilization of the plasma membrane in bovine, mouse, and porcine somatic cells. The permeabilized cells were treated with Xenopus laevis egg extracts. The treated cells showed exchange of nuclear proteins from extracts such as incorporation of Xenopus-specific histone B4 and Lamin LIII into nuclei. After resealing of the membrane, the cells showed upregulation of OCT4, SOX2, and NANOG expression. Our results suggest that reversible permeabilization with digitonin can be used to induce nuclear reprogramming and to activate pluripotent genes by a cell-free system.