Germline stem cells (GSCs) are known to transmit genetic information from parents to offspring. These GSCs can undergo reprogramming to transform themselves into pluripotent stem cells, called as Multipotent adult Germline stem cells (maGSCs). The mechanism of the reprogramming of GSCs to maGSCs is elusive. To investigate novel factors that may govern the process of reprogramming, the RNA-seq data of both GSCs and maGSCs were retrieved and subjected to Tuxedo protocol using Galaxy server. Total 1558 differentially expressed genes were identified from the analysis. Protein sequence in the FASTA format of all 1558 differentially expressed genes was retrieved and submitted to Pluripred web server to predict whether the proteins were pluripotent or not. A total of 232 proteins were predicted as pluripotent, and to identify the novel proteins, these were submitted to STRING database to obtain an interaction map. The obtained interaction map was submitted to Cytoscape, and various apps such as MCODE and Centiscape were used to identify the clusters and centrality measures between the nodes of the generated network. Five clusters were identified and ranked according to their score. Novel pluripotent proteins like cadherin related cdh5, cdh10 were predicted. Phox2b, Nrp2, Dll1, Shh, Gbx2, Nodal, Lefty1, Wnt7b, Pitx2, fgf4, Pou5f1, Nanog, Tet1, trim8, alx2, Dppa2, Prdm14,Sox11, Esrrb were predicted to be involved in the stem cell development. Dppa2, Sox11, Sox2, Bmp4, Shh, and Otp were predicted to be involved in positive regulation of the stem cell proliferation. Pathway analysis further revealed that signaling pathways such as Wnt, Jak-Stat and PI3K may play important role in the pluripotency of the maGSCs. Novel proteins involved in pluripotency, which were predicted by our findings, can be experimentally researched in future.

BACKGROUND

Embryo cryopreservation is the process that water is removed from the cell by cryoprotectant materials, and embryos are stored at temperature below zero. This process may affect the viability and developmental potential of embryos.

OBJECTIVE

In this study, the effect of the vitrification cryotop method on the expression level of Oct4 and Mest developmental genes in mouse blastocysts was examined.

METHODS

The collected 2-cell embryos of superovulated mouse by oviduct flushing were divided into non-vitrified and vitrified groups. These embryos were cultured to the blastocyst stage directly in the non-vitrified group and in the vitrified group, these embryos were cultured to 4-8 cell embryos, vitrified with cryotop in these stages and after 2-6 months, warmed and cultured to blastocyst embryos. Quantitative expression of two developmental genes, namely Oct4 and Mest, were performed in these groups, using RNA purification and Real-time RT-PCR.

RESULTS

Quantitative PCR analysis showed that the expression level of both genes, Oct4 and Mest, was reduced significantly in the vitrified-warmed group relative to the control group (p=0.046 and p=0.001).

CONCLUSIONS

This study revealed that morphologically normal embryos show a reduced amount of Oct4 and Mest transcripts which indicate that the vitrification method negatively effects the expression level of these two developmental genes.

OCT4, also known as POU5F1 (POU domain class 5 transcription factor 1), is a transcription factor that acts as a master regulator of pluripotency in embryonic stem cells and is one of the reprogramming factors required for generating induced pluripotent stem cells. The human OCT4 encodes three isoforms, OCT4A, OCT4B, and OCT4B1, which are generated by alternative splicing. Currently, the functions and expression patterns of OCT4B remain largely unknown in malignancies, especially in human glioblastomas. Here, we demonstrated the function of OCT4B in human glioblastomas. Among the isoform of OCT4B, OCT4B-190 (OCT4B^19kDa) was highly expressed in human glioblastoma stem cells and glioblastoma cells and was mainly detected in the cytoplasm rather than the nucleus. Overexpression of OCT4B^19kDa promoted colony formation of glioblastoma cells when grown in soft agar culture conditions. Clinical data analysis revealed that patients with gliomas that expressed OCT4B at high levels had a poorer prognosis than patients with gliomas that expressed OCT4B at low levels. Thus, OCT4B^19kDa may play a crucial role in regulating cancer cell survival and adaption in a rigid environment.

Mouse embryonic stem cells (mESC) and mouse epiblast stem cells (mEpiSC) share similar pluripotency factors like NANOG or POU5F1, however, their state of pluripotency differs significantly. mESC and mEpiSC can be derived from embryos generated by fertilization (FT) or by somatic cell nuclear transfer (NT). In this study we performed a 4-plex iTRAQ LC-MS/MS based approach, facilitating the multiplexed comparison of the four indicated types of stem cells. From four replicates of each cell type, 1650 proteins were quantified. 234 non redundant proteins with significant abundance alterations between FT/NT-mESC and FT/NT-mEpiSC, and 44 between FT and NT derived cells were detected. Bioinformatic analysis revealed that several pluripotency associated proteins, among them POU5F1, DNMT3L, TIF1B, and proteins involved in DNA repair like MSH2 and MSH6, are more abundant in mESC compared to mEpiSC. The abundance level of these proteins is not affected by the mode of embryo generation, whereas several cytoskeleton proteins show a higher abundance in NT-mESC compared to FT-mESC. In addition, a number of cytoskeletal proteins are enriched in mEpiSC, e.g., myosins, filamins and intermediate filament proteins, reflecting the progressed differentiation state of epiblast derived versus inner cell mass derived murine pluripotent stem cells.

UNASSIGNED

This study aims to get new insights in the pluripotency state of stem cells and to deepen the knowledge of early cell differentiation. In an iTRAQ MS approach, we quantitatively compared proteomes of inner cell mass derived stem cells (mESC) with epiblast derived stem cells (mEpiSC). These stem cell types are derived from embryos of different developmental stages, and therefore vary considerably in their state of pluripotency and reflect different stages of early differentiation. The proteins which show significant abundance differences between the two stem cell lines represent (i) promising targets to further decipher molecular processes during early embryo development and (ii) useful molecular markers to monitor early differentiation events of stem cells by targeted approaches.

Canine induced pluripotent stem cells (ciPSCs) are an attractive source for regenerative veterinary medicine, and may also serve as a disease model for human regenerative medicine. Extending the application of ciPSCs from bench to bedside, however, requires resolving many issues. We generated ciPSCs expressing doxycycline-inducible murine Oct3/4 (Pou5f1), Sox2, Klf4, and c-Myc, which were introduced using lentiviral vectors. The resultant ciPSCs required doxycycline to proliferate in the undifferentiated state. Those ciPSC colonies exhibiting basic fibroblast growth factor (bFGF)-dependent proliferation were dissociated into single cells for passaging, and were maintained on a Matrigel-coated dish without feeder cells in a serum-free medium. The established ciPSCs had the ability to differentiate into three germ layers, via formation of embryoid bodies, as well as into cells expressing the same markers as mesenchymal stem cells. These ciPSCs may thus serve as a suitable source of pluripotent stem cell lines for regenerative veterinary medicine, with fewer concerns of contamination from unknown animal components.

H19 is a maternally expressed, imprinted, noncoding RNA with tumor-suppressor activity. During mouse preimplantation development, H19 is primarily expressed in the trophectoderm cells. The purpose of this project was to determine allelic expression of H19 in pre- and peri-implantation mouse embryos. We were further interested in determining if loss of imprinted H19 expression during blastocyst development occurred as a result of superovulation and/or culture. Our last goal was to ascertain if differential H19 allelic expression occurred between the inner cell mass (ICM)-containing half and the primary trophoblast giant cell (PTGC)-containing half of the embryo. C57BL/6J((Cast-7))xC57BL/6J F1 embryos were collected from the uterus at 84, 96, and 108 h following natural ovulation or superovulation. In vitro-cultured F1 embryos were harvested from the oviduct at the 2-cell stage and cultured in KSOM + aa supplemented with amino acids or Whitten media and collected at the above-mentioned times. Allele-specific H19 expression in single embryos was determined by qRT-PCR followed by fluorescence resonance electron transfer or RT-PCR followed by restriction fragment length polymorphism and polyacrylamide gel electrophoresis (RFLP-PAGE). Peri-implantation embryos were microdissected into two sections, one containing the ICM and the other containing the PTGC. TaqMan probes for Dek, Pou5f1, Itga7, H19, and Igf2 were used to ascertain gene expression enrichment in each section. Allele-specific H19 expression in embryo sections was determined by RFLP-PAGE. We found that as embryos advance through preimplantation development they start expressing H19 in a biallelic manner and this phenomenon was observed in the cultured and the in vivo-developed embryos. The PTGC-containing half of the embryo had greater expression of H19 when compared to the ICM-containing half of the embryo, as determined by qRT-PCR. In conclusion, loss of imprinting of H19 occurs in the PTGC-containing section of peri-implantation mouse embryos. We speculate that this is part of a physiologic event at the time of implantation in the mouse.

Spatiotemporal expression of transcription factors is crucial for genomic reprogramming. Pou5f1 (Oct4) is an essential transcription factor for reprogramming. A recent study reported that OCT4A, which is crucial for establishment and maintenance of pluripotent cells, is expressed in oocytes, but maternal OCT4A is dispensable for totipotency induction. Whereas another study reported that OCT4B, which is not related to pluripotency, is predominantly expressed instead of OCT4A during early preimplantation phases in mice. To determine the expression states of OCT4 in murine preimplantation embryos, we conducted in-depth expression and functional analyses. We found that pluripotency-related OCT4 mainly localizes to the cytoplasm in early preimplantation phases, with no major nuclear localization until the 8-16-cell stage despite high expression in both oocytes and early embryos. RNA-sequencing analysis using oocytes and early preimplantation embryos could not identify the splice variants creating alternative forms of OCT4 protein. Forced expression of OCT4 in zygotes by the injection of polyadenylated mRNA clearly showed nuclear localization of OCT4 protein around 3-5-fold greater than physiological levels and impaired developmental competency in a dose-dependent manner. Embryos with modest overexpression of OCT4 could develop to the 16-cell stage; however, more than 50% of the embryos were arrested at this stage, similar to the results for OCT4 depletion. In contrast, extensive overexpression of OCT4 resulted in complete arrest at the 2-cell stage accompanied by downregulation of zygotically activated genes and repetitive elements related to the totipotent state. These results demonstrated that OCT4 protein localization was spatiotemporally altered during preimplantation development, and strict control of Oct4 protein levels was essential for proper totipotential reprogramming.

We previously reported that transcripts encoding the homeoprotein EGAM1N are expressed in preimplantation mouse embryos and embryonic stem (ES) cells, and the exogenous expression of EGAM1N inhibits the differentiation of ES cells. In order to clarify the relationship between the inhibition of differentiation and EGAM1N, we generated mouse MG1.19 ES cells stably expressing EGAM1N. Control transfectants with an empty vector formed relatively flattened cell colonies similar to those observed in parental MG1.19 cells. In contrast, Egam1n transfectants formed tightly aggregated cell colonies with increased localization of CDH1 at cell-to-cell interfaces. The protein levels of pluripotency factors, including TBX3 and SOX2, were also increased. The expression of Tbx3 transcripts was induced, although the level of Sox2 transcripts was almost unchanged. The expression of EGAM1N resulted in no obvious changes in the expression of genes encoding receptors, protein kinases, transcription factors, and their encoded proteins involved in the LIF-STAT3 signaling pathway. Alkaline phosphatase activity, a marker for the undifferentiated state, in Egam1n transfectants was exhibited in a clonal proliferation assay. When differentiation of Egam1n transfectants was induced, progression was prevented with increases in transcript levels of Pou5f1, Sox2, Nanog, Klf4, Tbx3, and their encoded proteins. However, Egam1n transfectants formed relatively flattened-cell layers as observed in the control, indicating that the expression of EGAM1N could not maintain LIF-independent self-renewal of ES cells. Overall, we suggest that expression of EGAM1N could inhibit differentiation, at least in part, by elevating the protein levels of pluripotency factors in MG1.19 ES cells.

UNASSIGNED

Pluripotent stem cells (PSCs) offer immense potential as a source for regenerative therapies. The teratoma assay is widely used in the field of stem cells and regenerative medicine, but the cell composition of teratoma is still elusive.

UNASSIGNED

We utilized PSCs expressing enhanced green fluorescent protein (EGFP) under the control of the Pou5f1 promoter to study the persistence of potential pluripotent cells during teratoma formation in vivo. OCT4-MES (mouse embryonic stem cells) were isolated from the blastocysts of 3.5-day OCT4-EGFP mice (transgenic mice express EGFP cDNA under the control of the Pou5f1 promoter) embryos, and TG iPS 1-7 (induced pluripotent stem cells) were generated from mouse embryonic fibroblasts (MEFs) from 13.5-day OCT4-EGFP mice embryos by infecting them with a virus carrying OCT4, SOX2, KLF4 and c-MYC. These pluripotent cells were characterized according to their morphology and expression of pluripotency markers. Their differentiation ability was studied with in vivo teratoma formation assays. Further differences between pluripotent cells were examined by real-time quantitative PCR (qPCR).

UNASSIGNED

The results showed that several OCT4-expressing PSCs escaped differentiation inside of teratomas, and these escaped cells (MES-FT, GFP-positive cells separated from OCT4-MES-derived teratomas; and iPS-FT, GFP-positive cells obtained from teratomas formed by TG iPS 1-7) retained their pluripotency. Interestingly, a small number of GFP-positive cells in teratomas formed by MES-FT and iPS-FT (MES-ST, GFP-positive cells isolated from MES-FT-derived teratomas; iPS-ST, GFP-positive cells obtained from teratomas formed by iPS-FT) were still pluripotent, as shown by alkaline phosphatase (AP) staining, immunofluorescent staining and PCR. MES-FT, iPS-FT, MES-ST and iPS-ST cells also expressed several markers associated with germ cell formation, such as Dazl, Stella and Stra8.

UNASSIGNED

In summary, a small number of PSCs escaped differentiation inside of teratomas, and these cells maintained pluripotency and partially developed towards germ cells. Both escaped PSCs and germ cells present a risk of tumor formation. Therefore, medical workers must be careful in preventing tumor formation when stem cells are used to treat specific diseases.

In the present study, we investigated whether precursor cells isolated from the dermis of infant human foreskin are capable to differentiate into hepatocyte-like cells upon sequential and gradual exposure to hepatogenic factors [fibroblast growth factor (FGF)-4, hepatocyte growth factor (HGF), insulin-transferrin-selenite (ITS), dexamethasone and oncostatin M (OSM)], mimicking the liver embryogenesis in vivo. Undifferentiated human skin-derived precursors (hSKP) are characterized by a fibroblast-like shape. Yet, they already express typical hepatic proteins, including cytokeratin (CK)-18, hepatocyte nuclear factor (HNF)-4 and HNF-1alpha. Microarray analysis further reveals gene expression of (i) the stemness markers nestin, POU5F1 (OCT-4), telomerase reverse transcriptase (TERT) and thymocyte differentiation antigen (THY)-1, (ii) biliary CK14 and CK19, (iii) biliary/foetal hepatic connexin (Cx)-43, and (iv) adult hepatic CK18, HNF-4 and HNF-1alpha. Upon differentiation, cells undergo morphological and phenotypic changes. As such, hSKP adopt a more polygonal-to-cuboidal cell shape. At the protein level, Cx43 expression is downregulated whereas typical hepatic markers, including alfa-foetoprotein (AFP), prealbumin (TTR) and albumin (ALB), become expressed in accordance to in vivo patterns observed during hepatogenesis. In conclusion, these data show for the first time that hSKP are capable to "trans" differentiate into hepatocyte-like cells upon mimicking the in vivo micro-environment of developing hepatocytes in vitro.

Vitamin C (Vc) has been widely studied in cell and embryo culture, and has recently been demonstrated to promote cellular reprogramming. The objective of this study was to identify a suitable Vc concentration that, when used to treat adult bovine fibroblasts serving as donor cells for nuclear transfer, improved donor-cell physiology and the developmental potential of the cloned embryos that the donor nuclei were used to create. A Vc concentration of 0.15 mM promoted cell proliferation and increased donor-cell 5-hydroxy methyl cytosine levels 2.73-fold (P < 0.05). The blastocyst rate was also significantly improved after nuclear transfer (39.6% treated vs. 26.0% control, P < 0.05); the average number of apoptotic cells in cloned blastocysts was significantly reduced (2.2 vs. 4.4, P < 0.05); and the inner cell mass-to-trophectoderm ratio (38.25% vs. 30.75%, P < 0.05) and expression of SOX2 (3.71-fold, P < 0.05) and POU5F1 (3.15-fold, P < 0.05) were significantly increased. These results suggested that Vc promotes cell proliferation, decreases DNA methylation levels in donor cells, and improves the developmental competence of bovine somatic-cell nuclear transfer embryos.

The homeoprotein EGAM1C was identified in preimplantation mouse embryos and embryonic stem (ES) cells. To explore the impact of EGAM1C on the hallmarks of mouse ES cells, MG1.19 cells stably expressing EGAM1C at levels similar to those in blastocysts were established using an episomal expression system. In the presence of leukemia inhibitory factor (+LIF), control transfectants with an empty vector formed flattened cell colonies, while Egam1c transfectants formed compacted colonies with increased E-CADHERIN expression. In Egam1c transfectants, the cellular contents of POU5F1 (OCT4), SOX2, TBX3, and NANOG increased. Cell growth was accelerated in an undifferentiated state sustained by LIF and in the course of differentiation. During clonal proliferation, EGAM1C stabilized the undifferentiated state. In adherent culture conditions, EGAM1C partly inhibited the progression of differentiation at least within a 4-day culture period in the presence of retinoic acid by preventing the downregulation of LIF signaling with a robust increase in TBX3 expression. Conversely, EGAM1C enhanced the expression of lineage marker genes Fgf5 (epiblast), T (mesoderm), Gata6 (primitive endoderm), and Cdx2 (trophectoderm) in -LIF conditions. In embryoid bodies expressing EGAM1C, the expression of marker genes for extraembryonic cell lineages, including Tpbpa (spongiotrophoblast) and Plat (parietal endoderm), increased. These results demonstrated that the ectopic expression of EGAM1C is capable of affecting the stabilization of an undifferentiated state and the progression of differentiation in MG1.19 ES cells, in addition to affecting cellular morphology and growth.

Embryonic stem cells typically show properties of long-term self-renewal and lack of differentiation. When appropriately stimulated, they are able to differentiate into all cell lineages, and lose their self-renewal characteristics. These properties are controlled by a series of genes encoding several transcription factors, including OCT4, the product of POU5F1 gene. OCT4 is expressed in germ cell tumors but also aberrantly in cancers developing in differentiated tissues. In a previous study, we observed a high expression of OCT4 in acute myeloid cell lines and primary cells, regardless of the acute myeloid leukemia (AML) subtype. In this study, we investigated the putative oncogenic role of OCT4 in proliferation and differentiation arrest. OCT4 expression was assessed in a panel of myeloid cell lines, together with clonogenic and proliferation properties, before and after differentiation in the presence of retinoic acid (RA). Same experiments were performed under short hairpin RNA (shRNA)-mediated OCT4 inhibition. In the presence of RA, we observed a decrease of OCT4 expression, associated with a loss of clonogenic and proliferation capacities, cell cycle arrest, and upregulation of p21, in HL60, NB4, KASUMI, and Me-1 cell lines. This effect was absent in the KG1a cell line, which did not differentiate. Downregulation of OCT4 by shRNA resulted in the same pattern of differentiation and loss of proliferation. Although KG1a did not differentiate, a decrease in proliferation was observed. Our findings suggest that OCT4 is implicated in the differentiation arrest at least in some types of AML, and that it also plays a role in cell proliferation through different oncogenic mechanisms. OCT4 might be a potential new target for antileukemic treatments.

The first lineage allocation during mouse development forms the trophectoderm and inner cell mass, in which Cdx2 and Pou5f1 display reciprocal expression. Yet Cdx2 is not required for trophectoderm specification in other mammals, such as the human, cow, pig, or in two marsupials, the tammar and opossum. The role of Cdx2 and Pou5f1 in the first lineage allocation of Sminthopsis macroura, the stripe-faced dunnart, is unknown. In this study, expression of Cdx2 and Pou5f1 during oogenesis, development from cleavage to blastocyst stages, and in the allocation of the first three lineages was analyzed for this dunnart. Cdx2 mRNA was present in late antral-stage oocytes, but not present again until Day 5.5. Pou5f1 mRNA was present from primary follicles to zygotes, and then expression resumed starting at the early unilaminar blastocyst stage. All cleavage stages and the pluriblast and trophoblast cells co-expressed CDX2 and POU5F1 proteins, which persisted until early stages of hypoblast formation. Hypoblast cells also show co-localisation of POU5F1 and CDX2 once they were allocated, and this persisted during their division and migration. Our studies suggest that CDX2, and possibly POU5F1, are maternal proteins, and that the first lineage to differentiate is the trophoblast, which differentiates to trophectoderm after shell loss one day before implantation. In the stripe-faced dunnart, cleavage cells, as well as trophoblast and pluriblast cells, are polarized, suggesting the continued presence of CDX2 in both lineages until late blastocyst stages may play a role in the formation and maintenance of polarity.

Lanosterol is a precursor of meiosis-activating sterols in the cholesterol biosynthetic pathway and induces a physiological signal that instructs the oocyte to reinitiate meiosis. In this study, we examined the effect of lanosterol on IVM of porcine oocytes, specifically on nuclear maturation, cytoplasmic maturation by investigating intracellular glutathione (GSH) levels and lipid content, embryonic development after parthenogenetic activation and somatic cell nuclear transfer (SCNT), and on gene expression in cumulus cells, oocytes, and SCNT-derived blastocysts. There was no significant difference in nuclear maturation rates between the control and treatment groups (10, 50, and 100 μM of lanosterol added to IVM culture medium). Supplementation with 50-μM lanosterol significantly increased lipid content and GSH levels and decreased reactive oxygen species levels compared with the control. In addition, oocytes treated with 50 μM of lanosterol exhibited significantly increased blastocyst formation rates and total cell numbers after parthenogenetic activation (30.3% and 63.9 vs. 21.6% and 36.5, respectively) and SCNT (18.2% and 53.7 vs. 12.6% and 37.5, respectively), when compared with the control group. Cumulus cells treated with 50 μM of lanosterol showed significantly increased 14α-demethylase, Δ14-reductase, and Δ7-reductase mRNA transcript levels. Significantly increased PPARγ, SREBF1, GPX1, and Bcl-2 and decreased Bax transcript levels were observed in mature oocytes treated with 50 μM of lanosterol compared with the control. SCNT blastocysts derived from 50-μM lanosterol-treated oocytes had significantly higher POU5F1, FGFR2, and Bcl-2 transcript levels than control SCNT-derived blastocysts. In conclusion, supplementation with 50 μM of lanosterol during IVM improves preimplantation development of SCNT embryos by elevating lipid content of oocytes, increasing GSH levels, decreasing reactive oxygen species levels, and regulating genes related to the cholesterol biosynthetic pathway in cumulus cells, to lipid metabolism and apoptosis in oocytes, and their developmental potential and apoptosis in blastocysts.

Appropriate epigenetic changes in preimplantation embryos are critical for embryonic development and successful pregnancy. The aim of this study was to evaluate the effects of some assisted reproductive techniques (ARTs) on a panel of epigenetic biomarkers by immunofluorescence staining at blastocyst stage. For this purpose, four treatment groups were designed: control (C), superovulation (S), superovulation+in vitro culture (SI), and superovulation+vitrification+in vitro culture (SVI). Results showed that vitrification decreased the developmental competence of embryos cultured in vitro (P<0.05). Semi-quantitative analysis revealed that vitrification decreased the fluorescence intensity of global DNA methylation in the inner cell mass (ICM), in SVI Group in comparison to C group (P<0.05). Superovulation, elevated the level of H3K9acetylation of trophectoderm (TE) in comparison to C and SI groups (P<0.05). Furthermore, ARTs manipulations influenced H3K9acetylation in the ICM (P<0.05). The fluorescence intensity of H4K12acetylation in TE for SVI group was higher than C and S (P<0.05). For H3K4tri-methylation, S group had higher fluorescence intensity in the ICM in comparison to SI and SVI (P<0.05). Finally, in vitro culture decreased Pou5f1 protein signal in comparison to in vivo-derived embryos at blastocyst stage (P<0.05). In conclusion, ART manipulations may have important influences on multiple epigenetic biomarkers.

Metastasis as a complex process involves loss of adhesion, migration, invasion and proliferation of cancer cells. Sulforaphane (SFN) is one of naturally occurring cancer chemopreventive isothiocyanates found in cruciferous vegetables, consumption of which has been associated with reduced risk of cancer. In this study, we describe effect of SFN on various aspects determining invasive behavior of MDA-MB-231 human breast carcinoma cells. We studied modulation of molecules associated with epithelial to mesenchymal transition (EMT), hypoxic marker CA IX and mitochondrially located peripheral benzodiazepine receptor (PBR) using flow cytometry, gene expression of matrix metalloproteinases MMP1, 3, 7, 9, 14, transcription factors POU5F1 and Twist1 mRNA by RT PCR, and cytokine production by multiplex bead assay. SFN downregulated PBR and vimentin expression in a dose dependent manner, but significantly affected neither HIF-1alpha, nor CA IX protein expression, nor VEGF and GLUT1 mRNA levels. Among studied MMPs, MMP7 and MMP14 mRNA were downregulated while no apparent effect on MMP1, MMP3 and MMP9 was observed. Further, we found significant down regulation of Twist1 and POU5F1, transcription factors that mediate EMT and the self-renewal of undifferentiated embryonic stem cells. SFN reduced also the production of pro-inflammatory cytokines IL-1beta, IL-6, TNF-alpha, IFN-gamma, immunomodulating cytokine IL-4 and growth factors involved in angiogenesis PDGF and VEGF. Our study shows that SFN efficacy is associated with the reversal of several biological characteristics connected with EMT or implicated in the matrix degradation and extracellular proteolysis, as well as with reduced production of pro-inflammatory cytokines and pro-angiogenic growth factors in MDA-MB-231 cells.

The objective of this study was to establish a versatile cell line for replication-incompetent virus production and inactivation with formaldehyde to generate a model of cell-based vaccine manufacturing process. To achieve this goal, we took advantage of the easily accessed chick embryonic fibroblasts. Nine-day old chick embryonic fibroblasts were obtained and subjected to be transduced with a set of lentivirus to develop a chick induced pluripotent stem (ciPS) cell line. Morphological features, positive periodic acid-Schiff staining as well as strong immunocytofluorescence of alkaline phosphatase, intestinal (ALPI) and POU class 5 homeobox 1 (POU5F1) proteins suggested that these chick embryonic fibroblasts have been transformed into ciPS cells. Further differentiation and immunocytofluorescence assays confirmed that this ciPS cell line possesses capacities and potentials to form embryoid bodies, differentiate into all three embryonic layers: ectoderm, mesoderm and endoderm with evidence of strongly positive and specific molecular markers. Immunoblot analysis next demonstrated that through recombinant DNA technology and the 2^nd generation lentiviral transfer system, the goose hemagglutinin gene (H5) gene was packaged into the replication-incompetent virus and highly expressed in a bladder cancer-derived cell line, T24, after transduction. The titer of ciPS-generated replication-incompetent virus is comparable to that from the Phoenix-AMPHO cell line, which is a commercial and high productive retrovirus producer. Our study successfully established a ciPS cell line which is able to produce replication-incompetent virus, providing a new strategy for cell-based vaccine production after virus inactivation.

The purpose of this study was to investigate the effects of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on transgene expression and development of porcine transgenic cloned embryos, specifically focusing on effects derived from TSA-treated donor cells or TSA-treated reconstructed embryos. The results showed that TSA treatment on reconstructed embryos modified the acetylation status, which significantly improved the development of porcine somatic cell nuclear transfer (SCNT) embryos in vitro, but not donor cells. Furthermore, the treatment of reconstructed embryos with TSA enhanced expression of the pluripotency-related gene POU5F1 and stimulated expression of the anti-apoptotic gene BCL-2. Enhanced green fluorescent protein (EGFP) mRNA expression of every group dropped drastically from donor cells to blastocysts. Interestingly, TSA is likely to prevent a decline in EGFP expression in nuclear reprogramming of porcine SCNT embryos. However DNA hypomethylation induced by modified histone acetylation of donor cells treated with TSA was significantly more effective in increasing EGFP expression in SCNT blastocysts. In conclusion, the acetylation status of both donor cells and reconstructed embryos modified by TSA treatment increased transgene expression and improved nuclear reprogramming and the developmental potential of porcine transgenic SCNT embryos.

Histone acetylation is one of the most important posttranslational modifications that contribute to transcriptional initiation and chromatin remodeling. In the present study, we aimed to investigate the effect of sodium butyrate (NaBu), a natural histone deacetylase inhibitor (HDACi), on the maturation of oocytes, preimplantation embryonic development, and expression of important developmental genes. The results indicated that NaBu decreased the rates of GVBD and the first polar body extrusion (PBE) in vitro in a dose-dependent manner. Meanwhile, NaBu treatment led to an abnormality in the spindle apparatus in oocytes in MI. However, the ratio of phosphor-extracellular signal-regulated kinases (p-ERK)/ERK significantly decreased in oocytes treated with 2.0 mM NaBu for 8 h. Furthermore, NaBu treatment at 2.0 mM improved the quality of embryos and the mRNA expression levels of important developmental genes such as HDAC1, Sox2, and Pou5f1. These data suggest that although a high concentration NaBu will impede the meiosis of oocytes, 2.0 mM NaBu will promote the development of embryos in vitro. Further investigation is needed to clarify the direct/indirect effects of NaBu on the regulation of important developmental genes and their subsequent impacts on full-term development in mammals.

Abnormal development and fetal loss during postimplantation period are concerns for production of nuclear transferred animals. Aberrant DNA methylation is one of the reasons for poor survival of cloned animals. In mammalian genome DNA, CpG islands are preferentially located at the start of transcription of housekeeping genes and are associated with tissue-specific genes. The correct and consecutive mechanisms of DNA methylation in the CpG islands are necessary for selective gene expressions that determine the properties of individual cells, tissues, and organs. In this study, we investigated the methylation status of the CpG islands of the bovine Leptin and POU5F1 genes in fetal and placental tissues from fetuses produced by artificial insemination (AI) and nuclear transfer (NT) at days 48 and 59 of pregnancy. Altered DNA methylation was observed in the normal and cloned fetal, placental, and endometrial tissues using bisulfite sequencing and pyrosequencing. Different tissue-specific methylated regions in the bovine Leptin and POU5F1 genes show a variable methylation status in NT fetuses compared to AI control.

Background: Current opinion suggests that expansion of cancer stem cells (CSCs) and activation of pro-tumoral inflammation cascade correlate with cancer progression. Materials and methods: We explored the possible contributions of MRC-5 cancer-associated fibroblasts to the expression profiles of CSC markers and inflammation-associated cell surface molecules. The liver cancer cell lines Bel-7402, SMMC-7721, MHCC-LM3, and HepG2 cultured in conditioned medium (CM) from MRC-5 served as test groups, whereas the liver cancer cell lines cultured in normal medium served as control groups. Results: Flow cytometry revealed that the proportions of CD90⁺ cells were significantly higher in MHCC-LM3-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, and moderately higher in Bel-7402-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, than in controls. The CD90⁺/CD45^- proportions were elevated in Bel-7402-(MRC-5)-CM and MHCC-LM3-(MRC-5)-CM cells, but reduced in HepG2-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, as compared to controls. Western blotting indicated that Nanog was downregulated in MHCC-LM3-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, compared to controls; that POU5F1 (OCT4/3) was downregulated in MHCC-LM3-(MRC-5)-CM, but upregulated in Bel-7402-(MRC-5)-CM and HepG2-(MRC-5)-CM cells, compared to controls, and that CK19 was upregulated in Bel-7402-(MRC-5)-CM and MHCC-LM3-(MRC-5)-CM cells, compared to controls. Proportions of cells expressing Toll-like receptor-1⁺ (TLR1) and TLR4 were significantly higher in MHCC-LM3-(MRC-5)-CM cells, and moderately higher in HepG2-(MRC-5)-CM cells, than controls. However, the TLR1⁺ and TLR4⁺ proportions were lower in Bel-7402-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells than controls. Proportions of CD25⁺ cells were reduced in HepG2-(MRC-5)-CM and SMMC-7721-(MRC-5)-CM cells, but elevated in MHCC-LM3-(MRC-5)-CM and Bel-7402-(MRC-5)-CM cells, compared to controls. Proportion of CD61⁺ cells was higher in liver cancer cells cultured in MRC-5-CM than in controls. Proportion of CD14⁺ cells was lower in HCC cells cultured in MRC-5-CM than in controls. Conclusion: MRC-5 extensively affected the production of CSC markers and inflammation-associated cell surface molecules. Tumor-targeting molecular therapies should consider these findings.

In present publication we describe for the first time the obtainment of cancer stem cells from a weakly metastatic human colorectal carcinoma cell line MIP101 via selecting from the native population the cells that express intensively an embryonic stem cell marker, POU5F1 (Oct4). We provide the evidence that these cells possess an elevated clonogenic and tumorigenic potential when compared to the native population, and this correlates to the hypothesis of cancer stem cells' primary role in the development of malignant neoplasms.