Epiblast stem cell subpopulations represent mouse embryos of distinct pregastrulation stages.

Embryonic stem cells (ESCs) comprise at least two populations of cells with divergent states of pluripotency. Here, we show that epiblast stem cells (EpiSCs) also comprise two distinct cell populations that can be distinguished by the expression of a specific Oct4-GFP marker. These two subpopulations, Oct4-GFP positive and negative EpiSCs, are capable of converting into each other in vitro. Oct4-GFP positive and negative EpiSCs are distinct from ESCs with respect to global gene expression pattern, epigenetic profile, and Oct4 enhancer utilization. Oct4-GFP negative cells share features with cells of the late mouse epiblast and cannot form chimeras. However, Oct4-GFP positive EpiSCs, which only represent a minor EpiSC fraction, resemble cells of the early epiblast and can readily contribute to chimeras. Our findings suggest that the rare ability of EpiSCs to contribute to chimeras is due to the presence of the minor EpiSC fraction representing the early epiblast.

Increased reprogramming capacity of mouse liver progenitor cells, compared with differentiated liver cells, requires the BAF complex.

BACKGROUND & AIMS: Ectopic expression of certain transcription factors can reprogram somatic cells to a pluripotent state. Hematopoietic and muscle stem cells can be more efficiently reprogrammed than differentiated blood or muscle cells, yet similar findings have not been shown in other primary organ systems. Moreover, molecular characteristics of the cellular hierarchy of tissues that influence reprogramming capacities need to be delineated. We analyzed the effect of differentiation stage of freshly isolated, mouse liver cells on the reprogramming efficiency. METHODS: Liver progenitor cell (LPC)-enriched cell fractions were isolated from adult (6-8 wk) and fetal (embryonic day 14.5) livers of mice and reprogrammed to become induced pluripotent stem (iPS) cells. Different transcription factors were expressed in liver cells, and markers of pluripotency were examined, along with the ability of iPS cells to differentiate, in vitro and in vivo, into different germ layers. RESULTS: Fetal and adult LPCs had significantly greater reprogramming efficiency after transduction with 3 or 4 reprogramming factors. Transduction efficiency-corrected reprogramming rates of fetal LPCs were 275-fold higher, compared with unsorted fetal liver cells, when 3 reprogramming factors were transduced. The increased reprogramming efficiency of LPCs, compared with differentiated liver cells, occurred independently of proliferation rates, but was associated with endogenous expression of reprogramming factors (Klf4 and c-Myc) and BAF (Brg1/Brm associated factor)-complex members Baf155 and Brg1, which mediate epigenetic changes during reprogramming. Knockdown of BAF complex members negated the increased reprogramming efficiency of LPCs, compared with non-LPCs. CONCLUSIONS: LPCs have intrinsic, cell proliferation-independent characteristics resulting in an increased reprogramming capacity compared to differentiated liver cells.

Conversion of mouse epiblast stem cells to an earlier pluripotency state by small molecules.

Epiblast stem cells (EpiSCs) are pluripotent cells derived from post-implantation late epiblasts in vitro. EpiSCs are incapable of contributing to chimerism, indicating that EpiSCs are less pluripotent and represent a later developmental pluripotency state compared with inner cell mass stage murine embryonic stem cells (mESCs). Using a chemical approach, we found that blockage of the TGFß pathway or inhibition of histone demethylase LSD1 with small molecule inhibitors induced dramatic morphological changes in EpiSCs toward mESC phenotypes with simultaneous activation of inner cell mass-specific gene expression. However, full conversion of EpiSCs to the mESC-like state with chimerism competence could be readily generated only with the combination of LSD1, ALK5, MEK, FGFR, and GSK3 inhibitors. Our results demonstrate that appropriate synergy of epigenetic and signaling modulations could convert cells at the later developmental pluripotency state to the earlier mESC-like pluripotency state, providing new insights into pluripotency regulation.

Generation of parthenogenetic induced pluripotent stem cells from parthenogenetic neural stem cells.

Somatic cells can achieve a pluripotent cell state in a process called pluripotential reprogramming. Multipotent stem cells can differentiate into cells of only one lineage, but pluripotent stem cells can give rise to cells of all three germ layers of an organism. In this study, we generated induced pluripotent stem (iPS) cells from bimaternal (uniparental) parthenogenetic neural stem cells (pNSCs) by transduction with either four (4F: Oct4, Klf4, Sox2, and c-Myc) or two (2F: Oct4 and Klf4) transcription factors. The resultant maternal iPS cells, which were reprogrammed directly from pNSCs, were capable of generating germ line-competent chimeras. Interestingly, analysis of global gene expression and imprinting status revealed that parthenogenetic iPS cells clustered closer to parthenogenetic ESCs than to female ESCs, with patterns that were clearly distinct from those of pNSCs.

Phosphorylation-dependent septin interaction of Bni5 is important for cytokinesis.

In budding yeast, septin plays as a scaffold to recruits protein components and regulates crucial cellular events including bud site selection, bud morphogenesis, Cdc28 activation pathway, and cytokinesis. Phosphorylation of Bni5 isolated as a suppressor for septin defect is essential to Swe1-dependent regulation of bud morphogenesis and mitotic entry. The mechanism by which Bni5 regulates normal septin function is not completely understood. Here, we provide evidence that Bni5 phosphorylation is important for interaction with septin component Cdc11 and for timely delocalization from septin filament at late mitosis. Phosphorylation-deficient bni5-4A was synthetically lethal with hof1Delta. bni5-4A cells had defective structure of septin ring and connected cell morphology, indicative of defects in cytokinesis. Two-hybrid analysis revealed that bni5-4A has a defect in direct interaction with Cdc11 and Cdc12. GFP-tagged bni5-4A was normally localized at mother-bud neck of budded cells before middle of mitosis. In contrast, at large-budded telophase cells, bni5-4A-GFP was defective in localization and disappeared from the neck approximately 2 min earlier than that of wild type, as evidenced by time-lapse analysis. Therefore, earlier delocalization of bni5-4A from septin filament is consistent with phosphorylation-dependent interaction with the septin component. These results suggest that timely delocalization of Bni5 by phosphorylation is important for septin function and regulation of cytokinesis.

Distinct Enhancer Activity of Oct4 in Naive and Primed Mouse Pluripotency.

Naive and primed pluripotent stem cells (PSCs) and germ cells express the Oct4 gene. The Oct4 gene contains two cis-regulatory elements, the distal enhancer (DE) and proximal enhancer (PE), which differentially control Oct4 expression in a cell-type-specific and stage-specific manner. Here, we generated double transgenic mice carrying both Oct4-ΔPE-GFP and Oct4-ΔDE-tdTomato (RFP), enabling us to simultaneously monitor the activity of DE and PE. Oct4 expression is stage-specifically regulated by DE and PE during embryonic and germ cell development. Using this dual reporter system, we successfully cultured pure populations of naive (GFP+RFP-) and primed (GFP-RFP+) PSCs. We found that GFP+RFP- cells were metastable (not naive) in serum-containing medium; stable naive pluripotent cells were observed in medium containing two inhibitors (Meki and GSKi) but lacked serum. Finally, we suggest that the activity of Oct4 DE and PE is regulated by the repressive histone marks and DNA methylation in a cell-type-specific manner.

VisuFect-mediated siRNA delivery into zygotes.

Current methods for delivering foreign genetic materials into mammalian cells are highly successful. However, these methods cannot be applied in oocyte or embryo systems due to their toxicity and low efficiency. Moreover, no satisfactory methods exist for delivering foreign genetic material without inducing physical damage to membranes. Here we developed an organic compound (VisuFect)-mediated small interfering RNA (siRNA) delivery method and evaluated this method in P19 cells and mouse zygotes. Oct4-siRNA conjugated VisuFect (Oct4-siRNA-VF) permeated the zona pellucida effectively and localized inside mouse zygotes without inducing membrane damage. Successful VisuFect-mediated delivery was further demonstrated by strong transcriptional repression of Oct4 expression by the delivered Oct4-siRNA, in addition to repressed embryonic development of mouse zygotes.

Establishment of a primed pluripotent epiblast stem cell in FGF4-based conditions.

Several mouse pluripotent stem cell types have been established either from mouse blastocysts and epiblasts. Among these, embryonic stem cells (ESCs) are considered to represent a "naïve", epiblast stem cells (EpiSCs) a "primed" pluripotent state. Although EpiSCs form derivatives of all three germ layers during in vitro differentiation, they rarely incorporate into the inner cell mass of blastocysts and rarely contribute to chimera formation following blastocyst injection. Here we successfully established homogeneous population of EpiSC lines with efficient chimera-forming capability using a medium containing fibroblast growth factor (FGF)-4. The expression levels of Rex1 and Nanog was very low although Oct4 level is comparable to ESCs. EpiSCs also expressed higher levels of epiblast markers, such as Cer1, Eomes, Fgf5, Sox17, and T, and further showed complete DNA methylation of Stella and Dppa5 promoters. However, the EpiSCs were clustered separately from E3 and T9 EpiSC lines and showed a completely different global gene expression pattern to ESCs. Furthermore, the EpiSCs were able to differentiate into all three germ layers in vitro and efficiently formed teratomas and chimeric embryos (21.4%) without germ-line contribution.

A color-tunable molecular beacon to sense miRNA-9 expression during neurogenesis.

A typical molecular beacon (MB) composing of a fluorophore and a quencher has been used to sense various intracellular biomolecules including microRNAs (miRNA, miR). However, the on/off-tunable miRNA MB is difficult to distinguish whether the observed low fluorescence brightness results from low miRNA expression or low transfection of the miRNA MB. We developed a color-tunable miRNA-9 MB (ColoR9 MB) to sense miR-9 expression-dependent color change. The ColoR9 MB was synthesized by a partially double-stranded DNA oligonucleotide containing a miR-9 binding site and a reporter probe with Cy3/black hole quencher 1 (BHQ1) at one end and a reference probe with Cy5.5 at the other end. The ColoR9 MB visualized CHO and P19 cells with red color in the absence of miR-9 and yellow color in the presence of miR-9. In vivo imaging demonstrated that the green fluorescence recovery of the reporter probe from the ColoR9 MB increased gradually during neuronal differentiation of P19 cells, whereas red fluorescence activity of the reference probe remained constant. These results showed the great specificity of sensing miR-9 expression- and neurogenesis-dependent color change.

Microinjection free delivery of miRNA inhibitor into zygotes.

The development of gene delivery systems into embryos is challenging due to technical difficulties, delivery efficiency and toxicity. Here, we developed an organic compound (VisuFect)-mediated gene delivery system for zygotes. The VisuFect, which is hydrophilic and Cy5.5-labeled, was conjugated with poly(A) oligo (VFA). The VFA into CHO cells showed clathrin-mediated internalization and no toxicity. The VFA successfully penetrated through the zona pellucida of fertilized eggs of various species including pigs, zebrafish, drosophilas and mice. The experiment with VisuFect-mediated delivery of the miR34c inhibitor showed similar results with direct microinjection of the miR34c inhibitor by suppressing the development of zygotes up to the blastocyst stage. Noticeable features of the VisuFect will provide great benefits for further studies on gene function in sperms and embryos.

Bioimaging of targeting cancers using aptamer-conjugated carbon nanodots.

Using the excellent emission properties of carbon nanodots, we have developed an aptamer-conjugated imaging probe for targeting cancers.

Transgenic chicken, mice, cattle, and pig embryos by somatic cell nuclear transfer into pig oocytes.

This study explored the possibility of producing transgenic cloned embryos by interspecies somatic cell nuclear transfer (iSCNT) of cattle, mice, and chicken donor cells into enucleated pig oocytes. Enhanced green florescent protein (EGFP)-expressing donor cells were used for the nuclear transfer. Results showed that the occurrence of first cleavage did not differ significantly when pig, cattle, mice, or chicken cells were used as donor nuclei (p>0.05). However, the rate of blastocyst formation was significantly higher in pig (14.9±2.1%; p<0.05) SCNT embryos than in cattle (6.3±2.5%), mice (4.2±1.4%), or chicken (5.1±2.4%) iSCNT embryos. The iSCNT embryos also contained a significantly less number of cells per blastocyst than those of SCNT pig embryos (p<0.05). All (100%) iSCNT embryos expressed the EGFP gene, as evidenced by the green florescence under ultraviolet (UV) illumination. Microinjection of purified mitochondria from cattle somatic cells into pig oocytes did not have any adverse effect on their postfertilization in vitro development and embryo quality (p>0.05). Moreover, NCSU23 medium, which was designed for in vitro culture of pig embryos, was able to support the in vitro development of cattle, mice, and chicken iSCNT embryos up to the blastocyst stage. Taken together, these data suggest that enucleated pig oocytes may be used as a universal cytoplast for production of transgenic cattle, mice, and chicken embryos by iSCNT. Furthermore, xenogenic transfer of mitochondria to the recipient cytoplast may not be the cause for poor embryonic development of cattle-pig iSCNT embryos.

Conserved and divergent roles of FGF signaling in mouse epiblast stem cells and human embryonic stem cells.

Mouse epiblast stem cells (EpiSCs) are cultured with FGF2 and Activin A, like human embryonic stem cells (hESCs), but the action of the associated pathways in EpiSCs has not been well characterized. Here, we show that activation of the Activin pathway promotes self-renewal of EpiSCs via direct activation of Nanog, whereas inhibition of this pathway induces neuroectodermal differentiation, like in hESCs. In contrast, the different roles of FGF signaling appear to be only partially conserved in the mouse. Our data suggest that FGF2 fails to cooperate with SMAD2/3 signaling in actively promoting EpiSC self-renewal through Nanog, in contrast to its role in hESCs. Rather, FGF appears to stabilize the epiblast state by dual inhibition of differentiation to neuroectoderm and of media-induced reversion to a mouse embryonic stem cell-like state. Our data extend the current model of cell fate decisions concerning EpiSCs by clarifying the distinct roles played by FGF signaling.

Cryopreservation of human embryos using ethylene glycol in controlled slow freezing.

BACKGROUND: Ethylene glycol (EG) has been successfully used as a cryoprotectant for vitrification of mammalian formula embryos (including human embryos) due to its low formula weight and high permeation into cells compared with other cryoprotectants, including propylene glycol (PROH). This study was carried out to evaluate the permeation and toxicity of EG and to investigate the effects of its use in a slow-freezing protocol on post-thaw development of mouse embryos and on pregnancy outcome of frozen human embryos. METHODS: Spare human embryos after embryo transfer were cryopreserved using 1.5 mol/l EG or PROH using a slow-freezing protocol which had been tested previously in mouse experiments. RESULTS: The post-thaw survival rate of human embryos in the EG group (80.6%) was significantly higher than that in the PROH group (65.2%, P < 0.05). The implantation and clinical pregnancy rates of human embryos in the EG group (20.3 and 46.9%) were significantly higher than those in the PROH group (7.5 and 24.6%, P < 0.05). CONCLUSIONS: Ethylene glycol may be a good substitute for PROH to cryopreserve human embryos using a slow-freezing protocol.