Mitochondrial and Metabolic Dynamics of Endometrial Stromal Cells During the Endometrial Cycle.

The endometrial cycle in response to hormonal stimulation is essential for implantation. The female has endometrium that repeats this cycle through about half of a lifetime. The cycle includes three phases, proliferative, secretory, and menstrual, and each phase has distinct characteristics. The endometrial stromal cells (EnSCs) in each phase also have specialized characteristics, including cell cycle, morphologies, and cellular metabolic state. So we hypothesized that the cells in each phase have unique mitochondrial morphologies because they are generally linked to cellular metabolic state. To investigate the metabolic characteristics in each phase, we investigated the mitochondrial morphologies by transmission electron microscopy, oxygen consumption rate (OCR), and intracellular adenosine triphosphate (ATP) production. The decidualized EnSCs have shorter mitochondria than those in the proliferative phase. Besides, they also displayed distinct intracellular structural characteristics compared with the proliferative phase, such as ribosome-rich endoplasmic reticulum and increased formation of vesicles. OCR and luminescent ATP detection assay revealed that the basal respiration and ATP production in the decidualized EnSCs were lower than those in the proliferative phase. Thus, we concluded that morphological and intracellular structural changes were induced during the decidualization. Moreover, the decreased mitochondrial length was shown to correlate with decreased dependency on oxidative phosphorylation and ATP concentration in EnSCs.

Alterations in intracellular Ca2+ levels in human endometrial stromal cells after decidualization.

Calcium (Ca2+) is an important element for many physiological functions of the uterus, including embryo implantation. Here, we investigated the possible involvement of altered intracellular Ca2+ levels in decidualization in human endometrial stromal cells (hEMSCs). hEMSCs showed high levels of mesenchymal stem cell marker expression (CD73, CD90, and CD105) and did not express markers of hematopoietic progenitor cells (CD31, CD34, CD45, and HLA-DR). Decidualization is a process of ovarian steroid-induced endometrial stromal cell proliferation and differentiation. Several types of ion channels, which are regulated by the ovarian hormones progesterone and estradiol, as well as growth factors, are important for endometrial receptivity and embryo implantation. The combined application of progesterone (1 µM medroxyprogesterone acetate) and cyclic AMP (0.5 mM) for 6 days not only elevated inositol 1,4,5-triphosphate receptor (IP3R)-mediated Ca2+ release and IP3R expression, it also promoted ORAI and STIM expression as well as cyclopiazonic acid-induced Ca2+ release. Finally, intracellular Ca2+ levels and ion channel gene expression influenced hEMSC proliferation. These results suggest that cytosolic Ca2+ dynamics, mediated by specific ion channels, serve as an important step in the decidualization of hEMSCs.

Predictive proteomic biomarkers for inflammatory bowel disease-associated cancer: where are we now in the era of the next generation proteomics?

Recent advances in genomic medicine have opened up the possibility of tailored medicine that may eventually replace traditional "one-size-fits all" approaches to the treatment of inflammatory bowel disease (IBD). In addition to exploring the interactions between hosts and microbes, referred to as the microbiome, a variety of strategies that can be tailored to an individual in the coming era of personalized medicine in the treatment of IBD are being investigated. These include prompt genomic screening of patients at risk of developing IBD, the utility of molecular discrimination of IBD subtypes among patients diagnosed with IBD, and the discovery of proteome biomarkers to diagnose or predict cancer risks. Host genetic factors influence the etiology of IBD, as do microbial ecosystems in the human bowel, which are not uniform, but instead represent many different microhabitats that can be influenced by diet and might affect processes essential to bowel metabolism. Further advances in basic research regarding intestinal inflammation may reveal new insights into the role of inflammatory mediators, referred to as the inflammasome, and the macromolecular complex of metabolites formed by intestinal bacteria. Collectively, knowledge of the inflammasome and metagenomics will lead to the development of biomarkers for IBD that target specific pathogenic mechanisms involved in the spontaneous progress of IBD. In this review article, our recent results regarding the discovery of potential proteomic biomarkers using a label-free quantification technique are introduced and on-going projects contributing to either the discrimination of IBD subtypes or to the prediction of cancer risks are accompanied by updated information from IBD biomarker research.

Apolipoprotein A-IV as a novel gene associated with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a common endocrine-metabolic disorder, affecting 6-10% of women of reproductive age. The etiology remains poorly understood. To investigate the differentially expressed proteins from PCOS patients versus healthy women, the protein expression in follicular fluid was analyzed using two-dimensional electrophoresis (2-DE). Since follicular fluid contains a number of secretory proteins required for oocyte fertilization and follicle maturation, it is possible that follicular fluid can be used as a provisional source for identifying pivotal proteins associated with PCOS. In this study, six overexpressed proteins [kininogen 1, cytokeratin 9, antithrombin, fibrinogen γ-chain, apolipoprotein A-IV (apoA-IV) precursor and α-1-B-glycoprotein (A1BG)] in follicular fluids from PCOS patients were identified with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) and nano LC-MS/MS. Western blot analysis confirmed that the protein expression levels of apoA-IV precursor and A1BG were increased in follicular fluid from PCOS patients compared with those from normal controls. The analysis of protein expression for other proteins revealed individual variation. These results facilitate the understanding of the molecular mechanisms of PCOS and provide candidate biomarkers for the development of diagnostic and therapeutic tools.

Genome-scale DNA methylation pattern profiling of human bone marrow mesenchymal stem cells in long-term culture.

Human bone marrow mesenchymal stem cells (MSCs) expanded in vitro exhibit not only a tendency to lose their proliferative potential, homing ability and telomere length but also genetic or epigenetic modifications, resulting in senescence. We compared differential methylation patterns of genes and miRNAs between early-passage [passage 5 (P5)] and late-passage (P15) cells and estimated the relationship between senescence and DNA methylation patterns. When we examined hypermethylated genes (methylation peak ≥ 2) at P5 or P15, 2,739 genes, including those related to fructose and mannose metabolism and calcium signaling pathways, and 2,587 genes, including those related to DNA replication, cell cycle and the PPAR signaling pathway, were hypermethylated at P5 and P15, respectively. There was common hypermethylation of 1,205 genes at both P5 and P15. In addition, genes that were hypermethylated at P5 (CPEB1, GMPPA, CDKN1A, TBX2, SMAD9 and MCM2) showed lower mRNA expression than did those hypermethylated at P15, whereas genes that were hypermethylated at P15 (MAML2, FEN1 and CDK4) showed lower mRNA expression than did those that were hypermethylated at P5, demonstrating that hypermethylation at DNA promoter regions inhibited gene expression and that hypomethylation increased gene expression. In the case of hypermethylation on miRNA, 27 miRNAs were hypermethylated at P5, whereas 44 miRNAs were hypermethylated at P15. These results show that hypermethylation increases at genes related to DNA replication, cell cycle and adipogenic differentiation due to long-term culture, which may in part affect MSC senescence.

Effects of acute ethanol treatment on NCCIT cells and NCCIT cell-derived embryoid bodies (EBs).

This study used human embryonic carcinoma (NCCIT) cells to evaluate genotoxicity and other effects of ethanol in earlier stages of cellular development. This undifferentiated pluripotent cell line has unlimited self-renewal capacity and has been shown to differentiate in vitro. We analyzed proteome expression profile of ethanol-treated NCCIT cells and NCCIT cell-derived embryoid bodies (EBs) by MALDI-TOF MS. To test the role of ethanol as an embryotoxic and/or teratogenic factor, MetaCore pathway analysis software (GeneGO) was used to evaluate the process of normal growth and differentiation of NCCIT cells and EBs. We compared the different protein expression profiles of ethanol-treated versus untreated NCCIT cells and NCCIT cell-derived EBs. The ethanol-treated NCCIT cells demonstrate significant up regulation of SMAP1, dual specificity phosphatase 1 and pro isomerase domain-containing 1, cytokeratin 18, triosephosphate isomerase and beta-tubulin. However, ethanol-treated NCCIT cell-derived EBs exhibited upregulated signatures of different proteins, including CDC25B phosphatase, alpha-enolase, 3-phosphoglycerate dehydrogenase and tumor suppressor patched L' isoform, which suggests that ethanol may play a different role in EBs. These proteins exert their function on transcriptional and translational processes. Moreover, the functional proteomic analysis confirms the relationship between ethanol and ethanol-regulated genes and various signaling pathways and networks. The data presented in this study contribute toward the understanding of the molecular mechanisms of ethanol in NCCIT cells and NCCIT cell-derived EBs.

High expression of large-conductance Ca2+-activated K+ channel in the CD133+ subpopulation of SH-SY5Y neuroblastoma cells.

Solid tumors contain a population of cancer stem cells (CSCs), and CD133 is widely used as a CSCs marker. We investigated the differences between CD133(+) and CD133(-) cells from the neuroblastoma cell line SH-SY5Y in terms of the expressions of voltage-gated ion channels. A CD133(+) enriched (>60%) population was isolated, and a subsequent whole-cell voltage-clamp study showed that these cells predominantly express TEA-sensitive outward K(+) currents (I(K,TEA)) and TTX-sensitive voltage-gated inward Na(+) currents (I(Na)). Cell-attached single channel recordings demonstrated higher density of large-conductance (155pS) channel in CD133(+) cells than in CD133(-) cells. The TEA-sensitivity and single channel conductance indicated the large-conductance Ca(2+)-activated K(+) channels (BK(Ca)). Furthermore, RT-PCR analysis of 22 transcripts of voltage-gated ion channels in SH-SY5Y cells showed the expressions of Cav1.3, Kir2.1, Kv1.4, Kv2.1, Kv4.2, Kv7.1, BK(Ca), and Nav1.7, and those of BK(Ca) and Nav1.7 were higher in CD133(+) than in CD133(-) cells. In addition, the increase of cytosolic Ca(2+) concentration ([Ca(2+)](c)) in response to ionomycin (a Ca(2+) ionophore) was higher and more sustained in CD133(+) than in CD133(-) cells. Plausibly membrane hyperpolarization via BK(Ca) might be responsible for the augmented Ca(2+) influx observed in CD133(+) cells. The physiological implications of the differential expression of BK(Ca) and Nav1.7 in CSCs require further investigation.

Human Hair Follicle Cells with the Cell Surface Marker CD34 Can Regenerate New Mouse Hair Follicles and Located in the Outer Root Sheath of Immunodeficient Nude Mice.

BACKGROUND AND OBJECTIVES: The bulge region of hair follicle has been reported as a putative reservoir of hair follicle stem cells. The purpose of this study was to compare hair follice CD34 negative (CD34-) cell with CD34 positive (CD34+) cell and to evaluate the ability to regenerate new hair of immunodeficient nude mouse. METHODS AND RESULTS: In this report, we isolated the cells with CD34, known as bulge-negative cell surface marker from cultured human hair follicle cells using by magnetic cell sorting (MACS), injected the cells to immunodeficient nude mouse. To determine immunological characterization, human hair follicle CD34+ cells and CD34- cells were assessed by flow cytometry. The localization of injected-CD34+ cells was assessed on formalin-fixed, paraffin-embedded mouse skin samples by in situ hybridization technique. Our findings show that the human hair follicle cells with cell surface marker CD34 were located in the outer root sheath of nude mouse after transplantation and the cells were able to regenerate new hair follicle in immunodeficient nude mouse. CD34- cells also were able to regenerate follicles in the mouse, however, CD34+ cells were able to regenerate much more hair follicle than CD34- cells. CONCLUSIONS: Therefore, the results of this study add new insight into the investigation of CD34 stem cell-related molecule in human hair follicles and suggest that not all human hair follicle stem cells reside in bulge region, but in a lager niche.

Obstetric outcome of patients with polycystic ovary syndrome treated by in vitro maturation and in vitro fertilization-embryo transfer.

OBJECTIVE: To assess the obstetric outcome of pregnancies resulting from in vitro maturation (IVM) and IVF-ET of immature oocytes retrieved from women with polycystic ovary syndrome (PCOS). DESIGN: Prospective observational study. SETTING: University fertility clinic. PATIENT(S): One hundred thirty-nine women undergoing 203 IVM treatment cycles. INTERVENTION(S): Immature oocyte recovery from unstimulated ovaries. In vitro oocyte maturation and fertilization. Fresh ET and assessment of obstetric outcomes in the pregnant women. MAIN OUTCOME MEASURE(S): Pregnancy and obstetric outcome. RESULT(S): Forty-one pregnancies were obtained in 187 ETs, resulting in a pregnancy rate of 21.9%. Except for three patients lost to follow-up in these pregnancies, the abortion and live birth rates were 36.8% (14 of 38) and 63.2% (24 of 38), respectively. The mean (+/-SD) gestational age and birth weight at delivery for singletons were 38.4 +/- 2.0 weeks (range, 33-41.6 weeks) and 3,252 +/- 516 g (1,750-4,100 g), respectively. For twins these were 36.7 +/- 1.9 weeks (34.6-39 weeks) and 2,361 +/- 304 g (1,900-2,990 g), respectively. Pregnancy complications occurred in five patients (13.2%); these included preterm labor (n = 3) and placenta previa (n = 2). Two patients (5.3%) had a major congenital anomaly diagnosed by ultrasonography. CONCLUSION(S): The abortion rate, gestational age and birth weight at delivery, and obstetric complications of pregnancies conceived by IVM-ET in women with PCOS were comparable with those of other women with PCOS being treated by conventional IVF-ET. In vitro maturation followed by IVF-ET seems to be a useful treatment option for women with PCOS, thus avoiding the risk of ovarian hyperstimulation syndrome.

Successful spouse pregnancy of male patients with severe aplastic anemia and chronic myelogenous leukemia using spermatozoa banked prior to bone marrow transplantation and using the ICSI procedure: case reports.

PURPOSE: To report two cases of successful spouse pregnancies which were conceived with long-term cryopreserved spermatozoa that had been collected prior to the male patients receiving a bone marrow transplant. METHODS: The first case is the pregnant wife of a 25-year-old man with chronic myelogenous leukemia, whose semen was collected before bone marrow transplant and then cryopreserved, thawed, and then injected into the wife's eggs via ICSI. The second case is a 28-year-old man with severe aplastic anemia who became a father after his wife's eggs were fertilized via ICSI with thawed spermatozoa. RESULTS: These two cases were achieved pregnancies. CONCLUSIONS: These cases support research that men with malignancy have the chance of fathering their own genetic children. Therefore, it is important to increase the awareness of clinicians, oncologists, and patients to the new developments in preserving fertility for cancer patients.