Increasing data implicate iron accumulation in tumorigenesis of the kidney, particularly the clear cell renal cell carcinoma (ccRCC) subtype. The von Hippel Lindau (VHL)/hypoxia inducible factor-α (HIF-α) axis is uniquely dysregulated in ccRCC and is a major regulator and regulatory target of iron metabolism, yet the role of iron in ccRCC tumorigenesis and its potential interplay with VHL inactivation remains unclear. We investigated whether ccRCC iron accumulation occurs due to increased cell dependency on iron for growth and survival as a result of VHL inactivation. Free iron levels were compared between four VHL-mutant ccRCC cell lines (786-0, A704, 769-P, RCC4) and two benign renal tubule epithelial cell lines (RPTEC, HRCEp) using the Phen Green SK fluorescent iron stain. Intracellular iron deprivation was achieved using two clinical iron chelator drugs, deferasirox (DFX) and deferoxamine (DFO), and chelator effects were measured on cell line growth, cell cycle phase, apoptosis, HIF-1α and HIF-2α protein levels and HIF-α transcriptional activity based on expression of target genes CA9, OCT4/POU5F1 and PDGFβ/PDGFB. Similar assays were performed in VHL-mutant ccRCC cells with and without ectopic wild-type VHL expression. Baseline free iron levels were significantly higher in ccRCC cell lines than benign renal cell lines. DFX depleted cellular free iron more rapidly than DFO and led to greater growth suppression of ccRCC cell lines (>90% at ~30-150 µM) than benign renal cell lines (~10-50% at up to 250 µM). Similar growth responses were observed using DFO, with the exception that a prolonged treatment duration was necessary to deplete cellular iron adequately for differential growth suppression of the less susceptible A704 ccRCC cell line relative to benign renal cell lines. Apoptosis and G1-phase cell cycle arrest were identified as potential mechanisms of chelator growth suppression based on their induction in ccRCC cell lines but not benign renal cell lines. Iron chelation in ccRCC cells but not benign renal cells suppressed HIF-1α and HIF-2α protein levels and transcriptional activity, and the degree and timing of HIF-2α suppression correlated with the onset of apoptosis. Restoration of wild-type VHL function in ccRCC cells was sufficient to prevent chelator-induced apoptosis and G1 cell cycle arrest, indicating that ccRCC susceptibility to iron deprivation is VHL inactivation-dependent. In conclusion, ccRCC cells are characterized by high free iron levels and a cancer-specific dependency on iron for HIF-α overexpression, cell cycle progression and apoptotic escape. This iron dependency is introduced by VHL inactivation, revealing a novel interplay between VHL/HIF-α dysregulation and ccRCC iron metabolism. Future study is warranted to determine if iron deprivation using chelator drugs provides an effective therapeutic strategy for targeting HIF-2α and suppressing tumor progression in ccRCC patients.

Nucleosomes form heterogeneous groups in vivo, named clutches. Clutches are smaller and less dense in mouse embryonic stem cells (ESCs) compared to neural progenitor cells (NPCs). Using coarse-grained modeling of the pluripotency Pou5f1 gene, we show that the genome-wide clutch differences between ESCs and NPCs can be reproduced at a single gene locus. Larger clutch formation in NPCs is associated with changes in the compaction and internucleosome contact probability of the Pou5f1 fiber. Using single-molecule tracking (SMT), we further show that the core histone protein H2B is dynamic, and its local mobility relates to the structural features of the chromatin fiber. H2B is less stable and explores larger areas in ESCs compared to NPCs. The amount of linker histone H1 critically affects local H2B dynamics. Our results have important implications for how nucleosome organization and H2B dynamics contribute to regulate gene activity and cell identity.

Keywords: chromatin dynamics; chromatin structure; mesoscale modeling; single molecule tracking.

The development of mammary gland as a lactogenic tissue is a highly coordinated multistep process. The epithelial cells of lactiferous tubules undergo profound changes during the developmental window of puberty, pregnancy, and lactation. Several hormones including estrogen, progesterone, glucocorticoids and prolactin act in concert, and orchestrate the development of mammary gland. Understanding the gene regulatory networks that coordinate proliferation and differentiation of HC11 Mammary Epithelial stem-like Cells (MEC) under the influence of lactogenic hormones is critical for elucidating the mechanism of lactogenesis in detail. In this study, we analyzed transcriptome profiles of undifferentiated MEC (normal) and compared them with Murine Embryonic Stem Cells (ESC) using next-generation mRNA sequencing. Further, we analyzed the transcriptome output during lactogenic differentiation of MEC following treatment with glucocorticoids (primed state) and both glucocorticoids and prolactin together (prolactin state). We established stage-specific gene regulatory networks in ESC and MEC (normal, priming and prolactin states). We validated the top up-and downregulated genes in each stage of differentiation of MEC by RT-PCR and found that they are comparable with that of RNA-seq data. HC11 MEC display decreased expression of Pou5f1 and Sox2, which is crucial for the differentiation of MEC, which otherwise ensure pluripotency to ESC. Cited4 is induced during priming and is involved in milk secretion. MEC upon exposure to both glucocorticoids and prolactin undergo terminal differentiation, which is associated with the expression of several genes, including Xbp1 and Cbp that are required for cell growth and differentiation. Our study also identified differential expression of transcription factors and epigenetic regulators in each stage of lactogenic differentiation. We also analyzed the transcriptome data for the pathways that are selectively activated during lactogenic differentiation. Further, we found that selective expression of chromatin modulators (Dnmt3l, Chd9) in response to glucocorticoids suggests a highly coordinated stage-specific lactogenic differentiation of MEC.

Oxidative stress is inevitable as it is derived from the handling, culturing, inherent metabolic activities, and medium supplementation of embryos. This study was performed to investigate the protective effect of chitosan nanoparticles (CNPs) on oxidative damage in porcine oocytes. For this purpose, cumulus-oocyte complexes (COCs) derived from porcine slaughterhouse ovaries were exposed to different concentrations of CNPs (0, 10, 25, and 50 ㎍/㎖) during in vitro maturation (IVM). Oocytes treated with 25 ㎍/㎖ CNPs showed significantly higher levels of GSH, along with a significant reduction in ROS levels compared to control, CNPs 10, and CNPs 50 groups. In parthenogenetic embryo production, the maturation rate was significantly higher in the CNPs25 group than that in the control and all other treated groups. In addition, when compared to the CNPs50 and control groups, CNPs25-treated oocytes showed significantly higher cleavage and blastocyst development rates. The highest concentration of CNPs reduced the total cell number and ratio of ICM: TE cells in parthenogenetic embryos, suggesting that there is a threshold where benefits are lost if exceeded. In cloned embryos, the CNPs25 group, as compared to all other treated groups, showed significantly higher maturation and cleavage rates. Furthermore, the blastocyst development rate in the CNPs25-treated group was significantly higher than that in the CNPs50-treated group, as was the total cell number. Moreover, we found that cloned embryos derived from the CNPs25-treated group showed significantly higher expression levels of Pou5f1, Dppa2, and Ndp52il genes, compared with those of the control and other treated groups. Our results demonstrated that 25 ㎍/㎖ CNPs treatment during IVM improves the developmental competence of porcine oocytes by reducing oxidative stress.

Keywords: chitosan nanoparticles; development; embryo; oxidative damage; porcine.

This study was performed to explore the effects of cobalamin treatment during in vitro maturation (IVM) of porcine oocytes. Specifically, the effects of cobalamin exposure on nuclear and cytoplasmic maturation of oocytes, diameter, glutathione (GSH) and reactive oxygen species (ROS) levels of matured oocytes, as well as development and gene expression of porcine parthenogenetic and cloned embryos were assessed. Cumulus-oocyte complexes were exposed to 200 pM cobalamin for 22 h or incubated for 22 h without cobalamin (controls). The mean diameter of cobalamin-treated oocytes was greater than that of control oocytes (160.0 vs. 154.5 μm; p < 0.05). GSH level increased but ROS level decreased in the cobalamin-treated oocyte group. Parthenogenetic embryos derived from cobalamin-treated oocytes showed improved oocyte maturation (91.3% vs. 83.8%), cleavage (88.9% vs. 82.1%), and blastocyst formation (38.7% vs. 31.9%) rates compared with control embryos (p < 0.05). Similarly, cloned embryos derived from cobalamin-treated oocytes showed higher oocyte maturation (89.2% vs. 82.2%), cleavage (87.5% vs. 80.3%), and blastocyst formation (30.0% vs. 23.8%) rates than control embryos (p < 0.05). Furthermore, in parthenogenetic and cloned embryos, total cell number, inner cell mass (ICM), trophectoderm (TE) expression, and ICM: TE ratio were higher in the cobalamin-treated group compared to that in the control group (p < 0.05). Cloned embryos in the cobalamin-treated group showed higher mRNA expression levels of POU5F1, DPPA2, and NDP52IL than control group embryos. Together, these results demonstrate that cobalamin treatment during IVM improves the developmental competence of porcine oocytes by neutralizing the free radicals produced in the IVM medium.

Keywords: Cobalamin; Development; Embryo; In vitro maturation; Porcine.

This study examined the effects of a caffeine treatment to improve nuclear reprogramming in porcine cloned embryos. Embryonic development and the expression of genes related to pluripotency (POU5F1, SOX2, NANOG, and CDX2) were compared after caffeine supplementation during manipulation at different concentrations (0, 1.25, 2.5, and 5.0 mM) and after varying the delayed activation time (control, 1, 2, and 4 h) after fusion. Caffeine added to media during manipulation produced a higher rate of development to blastocysts in the 1.25 mM group than in the other concentration groups (22.8% vs. 16.1%, 16.2%, and 19.2%; p < 0.05). When caffeine was added during the 4 h delayed activation, the 1.25 mM caffeine concentration produced a significantly higher rate of development than those in the other 4 h-activation-delayed caffeine concentration groups (22.4% vs. 9.4%, 14.0%, and 11.1%; p < 0.05). On the other hand, no significant improvement over that in the control group was observed when caffeine was supplemented during both the manipulation period and delayed activation period (16.0% vs. 15.2%), respectively. The levels of POU5F1, SOX2, and NANOG expression in blastocysts were significantly higher in the delayed activation caffeine group (4 h, 1.25 mM) than in the control group (1 h, 0 mM; p < 0.05). In conclusion, a caffeine treatment at 1.25 mM during delayed activation for 4 h can improve the preimplantation development of porcine somatic cell nuclear transfer embryos by activating nuclear reprogramming.

Testicular germ cell tumors (TGCTs) are classified into two main subtypes, seminoma (SE) and non-seminoma (NSE), but their molecular distinctions remain largely unexplored. Here, we used expression data for mRNAs and microRNAs (miRNAs) from The Cancer Genome Atlas (TCGA) to perform a systematic investigation to explain the different telomere length (TL) features between NSE (n = 48) and SE (n = 55). We found that TL elongation was dominant in NSE, whereas TL shortening prevailed in SE. We further showed that both mRNA and miRNA expression profiles could clearly distinguish these two subtypes. Notably, four telomere-related genes (TelGenes) showed significantly higher expression and positively correlated with telomere elongation in NSE than SE: three telomerase activity-related genes (TERT, WRAP53 and MYC) and an independent telomerase activity gene (ZSCAN4). We also found that the expression of genes encoding Yamanaka factors was positively correlated with telomere lengthening in NSE. Among them, SOX2 and MYC were highly expressed in NSE versus SE, while POU5F1 and KLF4 had the opposite patterns. These results suggested that enhanced expression of both TelGenes (TERT, WRAP53, MYC and ZSCAN4) and Yamanaka factors might induce telomere elongation in NSE. Conversely, the relative lack of telomerase activation and low expression of independent telomerase activity pathway during cell division may be contributed to telomere shortening in SE. Taken together, our results revealed the potential molecular profiles and regulatory roles involving the TL difference between NSE and SE, and provided a better molecular understanding of this complex disease.

Growth Differentiation Factor 8 (GDF8) is a member of the transforming growth factor-β (TGF-β) family and has been identified as a strong physiological regulator. This factor is expressed as a paracrine factor in mural granulosa cells. To investigate the effects of GDF8 on the in vitro maturation (IVM) of porcine oocytes, we assessed the quality of matured oocytes as well as the specific gene transcription and protein activation levels in oocytes and cumulus cells (CCs) after IVM and subsequent embryonic development after in vitro fertilization (IVF) and parthenogenetic activation (PA). Supplemental concentrations (0, 1, 10, and 100 ng/ml) of GDF8 were provided in IVM medium. Supplementation with GDF8 during IVM induced transcription of specific TGF-β receptor genes, such as ActRIIb and Alk4/5, and the recognition of the GDF8 by these receptors induced phosphorylation of p38 MAPK. Activated p38 MAPK signaling changed oocyte maturation and cumulus expansion-related gene transcription: Nrf2 and Bcl-2 in oocytes and PCNA, Nrf2, Has2, Ptx3, and TNFAIP6 in CCs. The altered gene expression pattern during IVM resulted in a 10% lower level of intracellular ROS in mature oocytes. The improved cytoplasmic maturation led to an increase in the fertilization efficiency and subsequent embryonic developmental competence. The embryonic development showed increases in the blastocyst formation rate and higher transcription levels of POU5F1 and BCL-2 in the blastocysts. The present study suggests that supplementation of GDF8 during IVM synergistically improved the developmental potential of IVF- and PA-derived porcine embryos by reducing the intracellular ROS level in oocytes by altering the transcription of specific genes and increasing the phosphorylation of p38 MAPK during IVM. In conclusion, for the first time, our results demonstrate that GDF8 can act as a paracrine factor to modulate oocyte maturation by regulating p38 MAPK phosphorylation and intracellular ROS level during porcine IVM.

Porcine somatic cell nuclear transfer (SCNT) is currently inefficient, as 1-3.95% of reconstructed embryos survive to term; inadequate or erroneous epigenetic reprogramming of the specialized donor somatic nucleus could be a primary reason. Therefore, a locus-specific analysis of DNA methylation dynamics in embryogenesis and the DNA methylation status of gametes and donor cells used for SCNT were conducted in the following developmentally important gene loci: POU5F1, NANOG, SOX2, H19, IGF2, IGF2R, XIST; and the retrotransposon LINE-1. There were significant epigenetic differences between the gametes and the somatic donor cells. Three gamete-specific differentially methylated regions (DMRs) in POU5F1, XIST, and LINE-1 were identified. A delayed demethylation process at POU5F1 and LINE-1 loci occurred after three successive cleavages, compared to the in vitro fertilized (IVF) embryos. Although cloned embryos could undergo de-methylation and re-methylation dynamics at the DMRs of imprinted genes (H19,IGF2R, and XIST), the re-methylation process was compromised, unlike in fertilized embryos. LINE-1 loci are widely dispersed across the whole genome, and LINE-1 DMR might be a potential porcine nuclear reprogramming epi-marker. Data from observations in our present and previous studies, and two published articles were pooled to produce a schematic diagram of locus-specific, DNA methylation dynamics of cloned and IVF embryos during porcine early embryogenesis. This also indicated aberrant DNA methylation reprogramming events, including inadequate DNA demethylation and insufficient re-methylation in cloned embryos. Further research should focus on mechanisms underlying demethylation during the early cleavage of embryos and de novo DNA methylation at the blastocyst stage.

Keywords: Cloned embryos; DNA methylation; Donor cells; In vitro fertilized embryos; Porcine.

Testicular germ cell cancer (TGCC) develops from pre-malignant germ neoplasia in situ (GCNIS) cells. GCNIS originates from fetal gonocytes (POU5F1⁺/MAGE-A4^-), which fail to differentiate to pre-spermatogonia (POU5F1^-/MAGE-A4⁺) and undergo malignant transformation. Gankyrin is an oncogene which has been shown to prevent POU5F1 degradation and specifically interact with MAGE-A4 in hepatocellular carcinoma (HCC) cells. We aimed to investigate the role of Gankyrin in progression from gonocyte to pre-invasive GCNIS and subsequent invasive TGCC.

We determined Gankyrin expression in human fetal testicular tissue (gestational weeks 9-20; n = 38), human adult testicular tissue with active spermatogenesis (n = 9), human testicular tissue with germ cell maturation delay (n = 4), testicular tissue from patients with pre-invasive GCNIS (n = 6), and invasive TGCC including seminoma (n = 6) and teratoma (n = 7). Functional analysis was performed in-vitro by siRNA knock-down of Gankyrin in the NTera2 cells (derived from embryonal carcinoma).Germ cell expression of Gankyrin was restricted to a sub-population of prespermatogonia in human fetal testes. Nuclear Gankyrin was also expressed in GCNIS cells of childhood and adult pre-invasive TGCC patients, and in GCNIS from seminoma and non-seminoma patients. Cytoplasmic expression was observed in seminoma tumour cells and NTera2 cells. Gankyrin knock-down in NTera2 cells resulted in an increase in apoptosis mediated via the TP53 pathway, whilst POU5F1 expression was unaffected. Furthermore, Gankyrin knock-down in NTera2 cells increased cisplatin sensitivity with an increase in cell death (13%, p < 0.05) following Gankyrin knock-down, when compared to cisplatin treatment alone, likely via BAX and FAS. Our results demonstrate that Gankyrin expression changes in germ cells during normal transition from gonocyte to prespermatogonia. In addition, changes in Gankyrin localisation are associated with progression of pre-invasive GCNIS to invasive TGCC. Furthermore, we found that Gankyrin is involved in the regulation of NTera2 cell survival and that a reduction in Gankyrin expression can modulate cisplatin sensitivity.These results suggest that manipulation of Gankyrin expression may reduce the cisplatin dose required for the treatment of TGCC, with benefits in reducing dose-dependent side effects of chemotherapy. Further studies are required in order to assess the effects of modulating Gankyrin on GCNIS/TGCC using in vivo models.

Mitochondria play a central role in the maintenance of the naive state of embryonic stem cells. Many details of the mechanism remain to be fully elucidated. Solute carrier family 25 member 36 (Slc25a36) might regulate mitochondrial function through transporting pyrimidine nucleotides for mtDNA/RNA synthesis. Its physical role in this process remains unknown; however, Slc25a36 was recently found to be highly expressed in naive mouse embryonic stem cells (mESCs). Here, the function of Slc25a36 was characterized as a maintenance factor of mESCs pluripotency. Slc25a36 deficiency (via knockdown) has been demonstrated to result in mitochondrial dysfunction, which induces the differentiation of mESCs. The expression of key pluripotency markers (Pou5f1, Sox2, Nanog, and Utf1) decreased, while that of key TE genes (Cdx2, Gata3, and Hand1) increased. Cdx2-positive cells emerged in Slc25a3Slc25a3Slc25a36, as well as the relationship of mitochondrial function with naive pluripotency maintenance and stem cell fate decision.

Adipose stem cells (ASCs) represent a reliable source of stem cells with a widely demonstrated potential in regenerative medicine and tissue engineering applications. New recent insights suggest that three-dimensional (3D) models may closely mimic the native tissue properties; spheroids from adipose derived stem cells (SASCs) exhibit enhanced regenerative abilities compared with those of 2D models. Stem cell therapy success is determined by "cell-quality"; for this reason, the involvement of stress signals and cellular aging need to be further investigated. Here, we performed a comparative analysis of genes connected with stemness, aging, telomeric length and oxidative stress, in 3D and 2D primary cultures. The expression levels of stemness-related markers and anti-aging Sirtuin1 were significantly up-regulated (P < 0.001) in SASCs-3D while gene expression of aging-related p16INK4a was increased in ASCs-2D (P < 0.001). The 3D and 2D cultures also had a different gene expression profile for genes related to telomere maintenance (Shelterin complex, RNA Binding proteins and DNA repair genes) (P < 0.01 and P < 0.001) and oxidative stress (aldehyde dehydrogenase class1 and 3) (P < 0.05, P < 0.01 and P < 0.001) and presented a striking large variation in their cellular redox state. Based on our findings, we propose a "cell quality" model of SASCs, highlighting a precise molecular expression of several genes involved with stemness (SOX2, POU5F1 and NANOG), anti-aging (SIRT1), oxidative stress (ALDH3) and telomeres maintenance.

Keywords: ALDH family; Adipose stem cells; Aging; Shelterin complex; Spheroid; Telomere length.

We previously demonstrated that a deficiency of natural antibodies against CD25, Mucin 1 (MUC1) and vascular endothelial growth factor receptor 1 (VEGFR1) could contribute to high risk of non-small cell lung cancer (NSCLC). This study was designed to investigate if natural IgG antibodies against POU domain class 5 transcription factor 1 (POU5F1), tumor necrosis factor-α (TNF-α) and the combination of CD25, VEGFR1 and MUC1 could play an anti-tumorigenic role against developing NSCLC. An ELISA was developed in-house to examine plasma IgG against peptide antigens derived from POU5F1, TNF-α and a combination of peptide antigens derived from CD25, MUC1 and VEGFR1 in 211 patients with NSCLC and 200 healthy controls. Mann-Whitney U test demonstrated that plasma IgG levels for the combination of peptide antigens derived from CD25, MUC1 and VEGFR1 were significantly lower in NSCLC patients than control subjects (Z=-12.978, P＜0.001) although plasma levels of IgG antibodies for POU5F1 and TNFα were not significantly changed. The in-house ELISA made with the CD25-MUC1-VEGFR1 combination had a sensitivity of 49.6% against a specificity of 95% to detect early stage NSCLC. In conclusion, natural antibodies against the combination of CD25, VEGFR1 and MUC1 may be an effective biomarker for early diagnosis of NSCLC.

Enzalutamide, an antiandrogen, is approved for therapy of castration resistant prostate cancer. Clinical applications have shown that approximately 30% of patients acquire resistance after a short period of treatment. However, the molecular mechanisms underlying this resistance is not completely understood. To identify transcriptomic signatures associated with acquisition of drug resistance we profiled gene expression of paired enzalutamide sensitive and resistant human prostate cancer LNCaP (lymph node carcinoma of the prostate) and C4-2B cells. Overlapping genes differentially regulated in the enzalutamide resistant cells were ranked by Ingenuity Pathway Analysis and their functional validation was performed using ingenuity knowledge database followed by confirmation to correlate transcript with protein expression. Analysis revealed that genes associated with cancer stem cells, such as POU5F1 (OCT4), SOX2, NANOG, BMI1, BMP2, CD44, SOX9, and ALDH1 were markedly upregulated in enzalutamide resistant cells. Amongst the pathways enriched in the enzalutamide-resistant cells were those associated with RUNX2, hedgehog, integrin signaling, and molecules associated with elastic fibers. Further examination of a patient cohort undergoing ADT and its comparison with no-ADT group demonstrated high expression of POU5F1 (OCT4), ALDH1, and SOX2 in ADT specimens, suggesting that they may be clinically relevant therapeutic targets. Altogether, our approach exhibits the potential of integrative transcriptomic analyses to identify critical genes and pathways of antiandrogen resistance as a promising approach for designing novel therapeutic strategies to circumvent drug resistance.

Keywords: antiandrogens; cancer stem cells; castrate resistant prostate cancer; enzalutamide resistance; transcriptional reprogramming.

The precise relationship between epigenetic perturbations and telomere dysfunction is an extant question. Previously, we showed that telomere dysfunction leads to differentiation instability in murine embryonic stem cells (mESCs) via perturbations in DNA methylation at pluripotency-factor promoters. Here, we uncovered that telomerase reverse transcriptase null (Tert^-/-) mESCs exhibit genome-wide perturbations in chromatin accessibility and gene expression during differentiation. These changes were accompanied by an increase of H3K27me3 globally, an altered chromatin landscape at the Pou5f1/Oct4 pluripotency gene promoter, and impaired Tert^-/- mESC differentiation. Inhibition of the Polycomb Repressive Complex 2 (PRC2), an H3K27 tri-methyltransferase, exacerbated the impairment in differentiation and pluripotency gene repression in Tert^-/- mESCs but not wild-type mESCs, whereas inhibition of H3K27me3 demethylation led to a partial rescue of the Tert^-/- phenotype. This data reveals a new interdependent relationship between H3K27me3 and telomere integrity in stem cell lineage commitment that may have implications in aging and cancer.

To study Thy1 as a fibroblast marker, SSEA1 as a marker of intermediate pluripotency, and Oct4 as a marker of established pluripotency in rat model of endometriosis.

In vivo animal study.

Endometriosis was induced in 20 albino female rats through autologous transplantation of one uterine horn to mesentery of intestine. Other 20 rats had their horn removed without transplantation (controls). Rats were sacrificed 4 weeks after induction surgery. Ectopic, eutopic, and control endometria were harvested from endometriosis and control animals respectively. Quantitative syber green based RT-PCR was used to detect expression of Thy-1 (CD90), FUT4 (SSEA1), and POU5F1 (Oct4) genes in tissues. Relative expression was normalized to that of β actin. Thy1, SSEA1, and Oct4 protein expression were detected by immunohistochemistry.

Ectopic endometrium expressed significantly higher mRNA of Oct4 and SSEA1 as compared to control endometrium. Expression levels of Oct4 and SSEA1 were comparable between ectopic and eutopic endometria and between eutopic and control endometria. Thy1 (CD90) gene expression level was comparable among ectopic, eutopic, and control endometria. Oct4 immunoscore were significantly higher in ectopic (6.6±0.91) than eutopic (2.5±0.78) or control endometrium (3.7±0.1) (P value 0.02). Thy1 and SSEA1 immunoscores were comparable among all three types of endometria.

Using rat model of endometriosis, ectopic endometrium showed significantly higher Oct4, and SSEA1, but similar Thy1 gene expression to that of control endometrium. This indicates increased transition from somatic to pluripotent cell states in ectopic endometrium which may play a role in endometriosis pathogenesis.

Aberrant expression of the pluripotency factor OCT4A in embryonal tumors of the central nervous system (CNS) is a key factor that contributes to tumor aggressiveness and correlates with poor patient survival. OCT4A overexpression has been shown to up-regulate miR-367, a microRNA (miRNA) that regulates pluripotency in embryonic stem cells and stem-like aggressive traits in cancer cells. Here, we show that (a) miR-367 is carried in microvesicles derived from embryonal CNS tumor cells expressing OCT4A; and (b) inhibition of miR-367 in these cells attenuates their aggressive traits. miR-367 silencing in OCT4A-overexpressing tumor cells significantly reduced their proliferative and invasive behavior, clonogenic activity, and tumorsphere generation capability. In vivo, targeting of miR-367 through direct injections of a specific inhibitor into the cerebrospinal fluid of Balb/C nude mice bearing OCT4A-overexpressing tumor xenografts inhibited tumor development and improved overall survival. miR-367 was also shown to target SUZ12, one of the core components of the polycomb repressive complex 2 known to be involved in epigenetic silencing of pluripotency-related genes, including POU5F1, which encodes OCT4A. Our findings reveal possible clinical applications of a cancer stemness pathway, highlighting miR-367 as a putative liquid biopsy biomarker that could be further explored to improve early diagnosis and prognosis prediction, and potentially serve as a therapeutic target in aggressive embryonal CNS tumors.

With an increasing focus on the large-scale expansion of mesenchymal stem cells (MSCs) required for clinical applications for the treatment of joint and bone diseases such as osteoarthritis, the optimisation of conditions for in vitro MSC expansion requires careful consideration to maintain native MSC characteristics. Physiological parameters such as oxygen concentration, media constituents, and passage numbers influence the properties of MSCs and may have major impact on their therapeutic potential. Cells grown under hypoxic conditions have been widely documented in clinical use. Culturing MSCs on large scale requires bioreactor culture; however, it is challenging to maintain low oxygen and other physiological parameters over several passages in large bioreactor vessels. The necessity to scale up the production of cells in vitro under normoxia may affect important attributes of MSCs. For these reasons, our study investigated the effects of normoxic and hypoxic culture condition on early- and late-passage adipose-derived MSCs. We examined effect of each condition on the expression of key stem cell marker genes POU5F1, NANOG, and KLF4, as well as differentiation genes RUNX2, COL1A1, SOX9, COL2A1, and PPARG. We found that expression levels of stem cell marker genes and osteogenic and chondrogenic genes were higher in normoxia compared to hypoxia. Furthermore, expression of these genes reduced with passage number, with the exception of PPARG, an adipose differentiation marker, possibly due to the adipose origin of the MSCs. We confirmed by flow cytometry the presence of cell surface markers CD105, CD73, and CD90 and lack of expression of CD45, CD34, CD14, and CD19 across all conditions. Furthermore, in vitro differentiation confirmed that both early- and late-passage adipose-derived MSCs grown in hypoxia or normoxia could differentiate into chondrogenic and osteogenic cell types. Our results demonstrate that the minimal standard criteria to define MSCs as suitable for laboratory-based and preclinical studies can be maintained in early- or late-passage MSCs cultured in hypoxia or normoxia. Therefore, any of these culture conditions could be used when scaling up MSCs in bioreactors for allogeneic clinical applications or tissue engineering for the treatment of joint and bone diseases such as osteoarthritis.

OBJECTIVE

Investigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT).

METHODS

Porcine embryos were produced by SCNT and cultured in the presence or absence of ascorbic acid. Ten-eleven translocation 3 (TET3) in oocytes was knocked down by siRNA injection. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, relative 5-methylcytosine and 5-hydroxymethylcytosine content in pronucleus were detected by realtime PCR.

RESULTS

Ascorbic acid significantly increased the development of porcine embryos produced by SCNT. After SCNT, transcript levels of reprogramming genes, Pou5f1, Sox2, and Klf were significantly increased in blastocysts. Furthermore, ascorbic acid reduced 5-methylcytosine content in pronuclear embryos compared with the control group. Knock down of TET3 in porcine oocytes significantly prevents the demethylation of somatic cell nucleus after SCNT, even if in the presence of ascorbic acid.

CONCLUSIONS

Ascorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus.

Oocytes from prepubertal animals have a reduced ability to undergo embryo development and produce viable offspring. The present work used an ovine model consisting of oocytes derived from adult and prepubertal donors to assess the molecular status of oocytes and preimplantation embryos with different developmental competence. The lower potential of oocytes of young donors was confirmed in terms of in vitro developmental capabilities and kinetics. A panel of genes including maternal effect (DPPA3, GDF9, NMP2, ZAR1) and housekeeping genes (ACTB, RPL19, SDHA, YWHAZ, ATP1A1), genes involved in DNA methylation (DNMT1, DNMT3A, DNMT3B), genomic imprinting (IGF2R), pluripotency (NANOG, POU5F1) and cell cycle regulation (CCNB1, CDK1, MELK) was relatively quantified. Temporal analysis during oocyte maturation and preimplantation embryo development evidenced patterns associated with donor age. With a few gene-specific exceptions, the differential model showed a reduced transcript abundance in immature prepubertal oocytes that completely reversed trend after fertilization, when higher mRNA levels were consistently observed in early embryos, indicating a delay in maternal transcript degradation. We propose that the molecular shortage in the prepubertal oocyte may affect its developmental potential and impair the early pathways of maternal mRNA clearance in the embryo. While confirming the different potential of oocytes derived from adult and prepubertal donors, our work showed for the first time a consistent delay in maternal transcript degradation in embryos derived from low competence oocytes that interestingly recalls the delayed developmental kinetics. Such abnormal transcript persistence may hinder further development and represents a novel perspective on the complexity of developmental competence.

Background: The prognostic and clinicopathological significance of POU Class 5 Homeobox 1 (POU5F1) among various cancers are disputable heretofore. The diagnostic value and functional mechanism of POU5F1 in liver hepatocellular carcinoma (LIHC) have not been studied thoroughly.

Methods: An integrative strategy of meta-analysis, bioinformatics, and wet-lab approach was used to explore the diagnostic and prognostic significance of POU5F1 in various types of tumors, especially in LIHC. Meta-analysis was utilized to investigate the impact of POU5F1 on prognosis and clinicopathological parameters in various cancers. The expression level and diagnostic value of POU5F1 were assessed by qPCR in plasma collected from LIHC patients and controls. The correlation between POU5F1 and tumor infiltrating immune cells (TIICs) in LIHC was evaluated by CIBERSORT. Gene set enrichment analysis (GSEA) was performed based on TCGA. Hub genes and related pathways were identified on the basis of co-expression genes of POU5F1.

Results: Elevated POU5F1 was associated with poor OS, DFS, RFS, and DSS in various cancers. POU5F1 was confirmed as an independent risk factor for LIHC and correlated with tumor occurrence, stage, and invasion depth. The combination of POU5F1 and AFP in plasma was with high diagnostic validity (AUC = 0.902, p < .001). Specifically, the level of POU5F1 was correlated with infiltrating levels of B cells, T cells, dendritic cells, and monocytes in LIHC. GSEA indicated that POU5F1 participated in multiple cancer-related pathways and cell proliferation pathways. Moreover, CBX3, CCHCR1, and NFYC were filtered as the central hub genes of POU5F1.

Conclusion: Our study identified POU5F1 as a pan-cancer gene that could not only be a prognostic and diagnostic biomarker in various cancers, especially in LIHC, but functionally carcinogenic in LIHC.

Keywords: LIHC; POU5F1; biomarker; immune infiltrates; pathogenesis.

The expression of specific developmentally important genes in preimplantation embryos is an accepted marker for unraveling the influence of single factors in studies that are mostly related to artificial reproduction techniques. Such studies, however, often reveal high levels of heterogeneity between single embryos, independently of the influence of factors of interest. A possible explanation for this variation could be the large variety of physiological and environmental factors to which early embryos are exposed and their ability to react to them. Here, we investigated several potentially important parameters of development at the same time, in blastocysts of the wild guinea pig (Cavia aperea) generated in vivo after natural mating. The optimal time for flushing fully developed blastocysts was between 123 and 126 hours after mating. The abundance of POU5F1 (P = 0.042), BAX (P < 0.001), SLC2A1 (P = 0.017), and DNMT3A (P < 0.001) mRNA changed significantly over time after mating. The number of sibling embryos present influenced STAT3 levels significantly (P = 0.02). Levels of BAX and POU5F1 were significantly affected by season (P = 0.03 and 0.04). The temporal pattern of SLC2A1 levels was significantly altered both after feeding a protein-deficient diet (P = 0.04) and temperature treatment (P = 0.04) of the sire. In addition, the identity of the father had a significant influence on POU5F1 (P = 0.049) and STAT3 (P < 0.001) mRNA abundances. These data report that the expression of specific genes in early embryos reflects the entire heterogeneity of their surroundings and that it is a plastic reaction toward a multifactorial environment.

This study aims to evaluate the deleterious effect of the mycotoxin aflatoxin B1 (AFB1) on bull spermatozoa and the carryver effect on the developing embryo. Proteomic analysis of AFB1-treated spermatozoa revealed differential expression of proteins associated with biological processes and cellular pathways that involved in spermatozoon function, fertilization competence and embryonic development. Therefore, we assume that factors delivered by the spermatozoa, regardless of DNA fragmentation, are also involved. To confirm this hypothesis, we have used the annexin V (AV) kit to separate the spermatozoa into apoptotic (AV+) and non-apoptotic (AV-) subpopulations which were found to correlate with high- and low DNA fragmentation, respectively. Fertilization with AV+ AFB1-treated spermatozoa, resulted in no blastocyst formation, whereas fertilization with AV- spermatozoa resulted in reduced cleavage rate and formation of genetically altered blastocysts (POU5F1 and SOX2). Microarray analysis of blastocysts derived from 10 µM AFB1-treated spermatozoa revealed differential expression of 345 genes that involved in cellular pathways such as embryo and placenta development, cell cycle, DNA repair and histone modification, and in signaling pathways, especially calcium signaling pathway. This is the first report on deleterious carrying over effects of AFB1 from the bovine spermatozoa to the formed embryo. Our findings suggest that aside from the damage caused by AFB1 to spermatozoa's DNA integrity, additional damage mechanisms are involved.