Ferroportin expression and regulation in human placenta/fetal membranes: Implications for ferroptosis and adverse pregnancy outcomes.

Iron overload is associated with pregnancy complications. Ferroportin (FPN) is the only known iron exporter in mammalian cells. We hypothesize that FPN is functionally important in ferrotopsis, a process of iron-dependent non-apoptotic programmed cell death, and may have a critical role to play in pregnancy success. We investigated the expression of FPN in placenta/fetal membranes by immunohistochemistry in tissues collected from pregnancies with/without preeclampsia (PE) and spontaneous preterm birth (SPTB). FPN was highly expressed in both trophoblasts and decidual cells found in placenta/fetal membranes. Staining was significantly reduced in fetal membranes from SPTB versus healthy pregnancies (P = 0.046). FPN expression in immortalized human endometrial stromal cells (HESC) increased with in vitro decidualization induction using 1 µM of medroxyprogesterone acetate and 0.5 mM of dibutyryl-cAMP. In addition, both HESC cells and immortalized extravillous trophoblast SW71 cells with FPN knockdown showed significant sensitivity to ferroptosis inducer, erastin (P < 0.001 and P = 0.009, respectively). The survival of both HESC and SW71 cells was not negatively affected by iron supplementation with ferric ammonium citrate in the medium. However, SW71 cells were more sensitive than HESC cells to physiologic iron in the presence of a non-lethal dose of erastin (P < 0.001). Taken together, our data demonstrating increased sensitivity of FPN knockdown HESC and SW71 cells to erastin and increased sensitivity of trophoblasts to iron overload under ferroptotic stress support the hypothesis that FPN protects against ferroptosis during pregnancy.

Characterization of miR-200 family members as blood biomarkers for human and laying hen ovarian cancer.

MicroRNA-200 (miR-200) family is highly expressed in ovarian cancer. We evaluated the levels of family members relative to the internal control miR-103a in ovarian cancer and control blood specimens collected from American and Hong Kong Chinese institutions, as well as from a laying hen spontaneous ovarian cancer model. The levels of miR-200a, miR-200b and miR-200c were significantly elevated in all human cancer versus all control blood samples. Further analyses showed significantly higher miR-200 levels in Chinese control (except miR-429) and cancer (except miR-200a and miR141) samples than their respective American counterparts. Subtype-specific analysis showed that miR-200b had an overall elevated level in serous cancer compared with controls, whereas miR-429 was significantly elevated in clear cell and endometrioid cancer versus controls. MiR-429 was also significantly elevated in cancer versus control in laying hen plasma samples, consistent with the fact that endometrioid tumor is the prevalent type in this species. A neural network model consisting of miR-200a/200b/429/141 showed an area under the curve (AUC) value of 0.904 for American ovarian cancer prediction, whereas a model consisting of miR-200b/200c/429/141 showed an AUC value of 0.901 for Chinese women. Hence, miR-200 is informative as blood biomarkers for both human and laying hen ovarian cancer.

Endometrial Decidualization: The Primary Driver of Pregnancy Health.

Interventions to prevent pregnancy complications have been largely unsuccessful. We suggest this is because the foundation for a healthy pregnancy is laid prior to the establishment of the pregnancy at the time of endometrial decidualization. Humans are one of only a few mammalian viviparous species in which decidualization begins during the latter half of each menstrual cycle and is therefore independent of the conceptus. Failure to adequately prepare (decidualize) the endometrium hormonally, biochemically, and immunologically in anticipation of the approaching blastocyst-including the downregulation of genes involved in the pro- inflammatory response and resisting tissue invasion along with the increased expression of genes that promote angiogenesis, foster immune tolerance, and facilitate tissue invasion-leads to abnormal implantation/placentation and ultimately to adverse pregnancy outcome. We hypothesize, therefore, that the primary driver of pregnancy health is the quality of the soil, not the seed.

MicroRNA-200 family governs ovarian inclusion cyst formation and mode of ovarian cancer spread.

Epidemiologic and histopathologic findings and the laying hen model support the long-standing incessant ovulation hypothesis and cortical inclusion cyst involvement in sporadic ovarian cancer development. MicroRNA-200 (miR-200) family is highly expressed in ovarian cancer. Herewith, we show that ovarian surface epithelial (OSE) cells with ectopic miR-200 expression formed stabilized cysts in three-dimensional (3D) organotypic culture with E-cadherin fragment expression and steroid hormone pathway activation, whereas ovarian cancer 3D cultures with miR-200 knockdown showed elevated TGF-ß expression, mitotic spindle disorientation, increased lumenization, disruption of ROCK-mediated myosin II phosphorylation, and SRC signaling, which led to histotype-dependent loss of collective movement in tumor spread. Gene expression profiling revealed that epithelial-mesenchymal transition and hypoxia were the top enriched gene sets regulated by miR-200 in both OSE and ovarian cancer cells. The molecular changes uncovered by the in vitro studies were verified in both human and laying hen ovarian cysts and tumor specimens. As miR-200 is also essential for ovulation, our results of estrogen pathway activation in miR-200-expressing OSE cells add another intriguing link between incessant ovulation and ovarian carcinogenesis.

Endometriosis: The Role of Iron Overload and Ferroptosis.

Iron is an essential element for cell survival, and iron deficiency is a known risk factor for many reproductive disorders. Paradoxically, such disorders are also seen more commonly under conditions of iron excess. Here, we focus on the problem of iron overload in women's health, using endometriosis as a model system. We propose (i) that a primary defect in endometriosis is abnormal eutopic endometrium characterized by resistance to ferroptosis, a process of iron-mediated non-apoptotic programmed cell death, which allows cells spread via retrograde menstruation to survive, implant, and establish endometriotic lesions within the abdominal cavity, and (ii) that dysregulated iron homeostasis may be critical to the subsequent pathophysiology of endometriotic lesions with localized iron overload and inflammation. We further investigate the association between endometriosis and hypercholesterolemia and suggest that an interaction between the mevalonate cholesterol biosynthetic pathway and ferroptosis signaling may provide a molecular basis to explain how it is that, in some women, endometrial tissues survive and thrive under ferroptotic pressure, colonize at ectopic sites, and expand into endometriotic lesions.

The Impact of Iron Overload and Ferroptosis on Reproductive Disorders in Humans: Implications for Preeclampsia.

Iron is an essential element for the survival of most organisms, including humans. Demand for iron increases significantly during pregnancy to support growth and development of the fetus. Paradoxically, epidemiologic studies have shown that excessive iron intake and/or high iron status can be detrimental to pregnancy and is associated with reproductive disorders ranging from endometriosis to preeclampsia. Reproductive complications resulting from iron deficiency have been reviewed elsewhere. Here, we focus on reproductive disorders associated with iron overload and the contribution of ferroptosis-programmed cell death mediated by iron-dependent lipid peroxidation within cell membranes-using preeclampsia as a model system. We propose that the clinical expressions of many reproductive disorders and pregnancy complications may be due to an underlying ferroptopathy (elemental iron-associated disease), characterized by a dysregulation in iron homeostasis leading to excessive ferroptosis.

Comparison of benign peritoneal fluid- and ovarian cancer ascites-derived extracellular vesicle RNA biomarkers.

BACKGROUND: Extracellular vesicles (EVs) are considered as a new class of resources for potential biomarkers. We analyzed expression of specific mRNA and miRNA in EVs derived from ovarian cancer ascites and the ideal controls, peritoneal fluids from benign patients for potential early detection and prognostic biomarkers. METHODS: Fluids were collected from subjects with benign cysts or endometrioma (n = 10), or low/high grade serous ovarian carcinoma (n = 8). EV particles were captured using primarily ExoComplete filterplate or ultracentrifugation and analyzed by nanoparticle tracking analysis, ELISA, and scanning electron microscopy. EV RNAs extracted from two ascites and three peritoneal fluids were submitted for next-generation sequencing. The expression of 34 mRNA and 18 miRNAs in the EVs isolated from patient fluids and cell line media was determined using qPCR. RESULTS: EVs isolated from patient samples had concentrations greater than 1010 EV particles/mL and 30% were EpCAM-positive based on ELISA. EV particle sizes averaged 113 ± 11.5 nm. The qPCR studies identified five mRNA (CA11, MEDAG, LAMA4, SPINT2, NANOG) and six miRNA (let-7b, miR23b, miR29a, miR30d, miR205, miR720) that were significantly differentially expressed between cancer ascites and peritoneal fluids. In addition, CA11 mRNA was decreased to 0.5-fold and SPINT2 and NANOG mRNA were significantly increased up to 100-fold in conditioned media of cancer cells compared to immortalized ovarian surface and fallopian tube epithelial cell lines, the hypothesized cells of origin for ovarian cancer development. CONCLUSIONS: This study indicates that EV mRNA profiles can reflect the disease stage and may provide a potentially novel source for discovery of biomarkers in ovarian cancer.

Progesterone Inhibits Apoptosis in Fetal Membranes by Altering Expression of Both Pro- and Antiapoptotic Proteins.

OBJECTIVE: Progesterone supplementation prevents preterm birth (PTB) in some high-risk women, but its mechanism of action is unknown. One-third of PTB is associated with preterm premature rupture of membranes (PPROMs). We have previously shown that progesterone inhibits basal and Tumor Necrosis Factor (TNF) α-induced apoptosis in an explant model of human fetal membranes. This study investigates the molecular mechanisms responsible for progesterone-mediated inhibition of apoptosis in fetal membranes. METHODS: Human fetal membranes were collected at elective cesarean at term (no labor, no infection; n = 6), washed, and pretreated with/without progesterone (125 ng/mL) for 24 hours. Thereafter, membranes were treated with/without TNFα (50 ng/mL) and/or progesterone for 48 hours, harvested, and homogenized. Apoptosis was determined by evaluating caspase-3, -8, and -9 activities. Expression of pro- BH3 interacting domain death against, Bc1-2 associated X protein (BID, BAX) and antiapoptotic proteins (X-linked inhibitor of apoptosis protein [XIAP], Bcl-2, FLICE inhibitory protein [FLIP]) were measured by Western blot. RESULTS: TNFα increased apoptosis (measured by caspase-3, -8, and -9 activities) in fetal membranes, and this effect was abrogated by progesterone. Under basal conditions, progesterone suppressed expression of the proapoptotic protein, BID, by 0.45 (0.14)-fold, and increased expression of the antiapoptotic proteins, Bcl-2 and XIAP; no change was seen in BAX or FLIP. In contrast, TNFα increased BID expression by 5.15 (2.92)-fold, which was prevented by pretreatment with progesterone. CONCLUSIONS: Progesterone inhibits apoptosis in fetal membranes by suppressing expression of the proapoptotic protein, BID (for both basal and TNFα-induced apoptosis), and upregulating expression of the antiapoptotic proteins, XIAP and Bcl-2 (under basal conditions only). These data provide a mechanism by which progesterone supplementation may prevent PPROM and PTB in some women at high risk.

Filter-Based Extracellular Vesicle mRNA Isolation and High-Throughput Gene Expression Analysis.

Extracellular vesicles (EVs) are a heterogeneous group of membrane-encapsulated particles with different ranges of size, density, and cargo. Various types of RNA including mRNA are enclosed within EVs and can serve as novel biomarkers for disease detection and patient management. Ultracentrifugation, precipitation , antibody-based capture and filter-based methods are available as in-house laboratory procedures or commercially available kits to isolate EVs. Here, we describe a filter-based method for EV mRNA isolation that is designed for parallel processing of large sample numbers.

Characterization of MicroRNA-200 pathway in ovarian cancer and serous intraepithelial carcinoma of fallopian tube.

BACKGROUND: Ovarian cancer is the leading cause of death among gynecologic diseases in Western countries. We have previously identified a miR-200-E-cadherin axis that plays an important role in ovarian inclusion cyst formation and tumor invasion. The purpose of this study was to determine if the miR-200 pathway is involved in the early stages of ovarian cancer pathogenesis by studying the expression levels of the pathway components in a panel of clinical ovarian tissues, and fallopian tube tissues harboring serous tubal intraepithelial carcinomas (STICs), a suggested precursor lesion for high-grade serous tumors. METHODS: RNA prepared from ovarian and fallopian tube epithelial and stromal fibroblasts was subjected to quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) to determine the expression of miR-200 families, target and effector genes and analyzed for clinical association. The effects of exogenous miR-200 on marker expression in normal cells were determined by qRT-PCR and fluorescence imaging after transfection of miR-200 precursors. RESULTS: Ovarian epithelial tumor cells showed concurrent up-regulation of miR-200, down-regulation of the four target genes (ZEB1, ZEB2, TGFß1 and TGFß2), and up-regulation of effector genes that were negatively regulated by the target genes. STIC tumor cells showed a similar trend of expression patterns, although the effects did not reach significance because of small sample sizes. Transfection of synthetic miR-200 precursors into normal ovarian surface epithelial (OSE) and fallopian tube epithelial (FTE) cells confirmed reduced expression of the target genes and elevated levels of the effector genes CDH1, CRB3 and EpCAM in both normal OSE and FTE cells. However, only FTE cells had a specific induction of CA125 after miR-200 precursor transfection. CONCLUSIONS: The activation of the miR-200 pathway may be an early event that renders the OSE and FTE cells more susceptible to oncogenic mutations and histologic differentiation. As high-grade serous ovarian carcinomas (HGSOC) usually express high levels of CA125, the induction of CA125 expression in FTE cells by miR-200 precursor transfection is consistent with the notion that HGSOC has an origin in the distal fallopian tube.

The Functions of MicroRNA-200 Family in Ovarian Cancer: Beyond Epithelial-Mesenchymal Transition.

The majority of studies on microRNA-200 family members (miR-200s) in human cancers are based on the premise that miR-200s maintain epithelial cell integrity by suppressing epithelial-mesenchymal transition (EMT) through direct inhibition of mesenchymal transcription factors zinc finger E-box-binding homeobox 1/2 (ZEB1/ZEB2) and transforming growth factor-ß (TGF-ß), a potent inducer of EMT. Hence, downregulation of miR-200 in cancer cells promotes EMT and cancer metastasis. Yet, miR-200s are highly expressed in ovarian cancer, and ovarian cancer metastasizes primarily by dissemination within the pelvic cavity. In this review, we will refocus the epithelial property of ovarian cancer cells and the role of miR-200s in safeguarding this property, as well as the diverse roles of miR-200s in inclusion cyst formation, cancer cell growth, collective movement, angiogenesis, exosome-mediated cell communication, and chemoresponse. Taken together, miR-200s play a significant role in the initiation, progression and metastasis of ovarian cancer and may serve as diagnostic biomarkers and a target in therapeutic development.

Cyclin A1 expression and paclitaxel resistance in human ovarian cancer cells.

BACKGROUND: The development of intrinsic and acquired resistance to antineoplastic agents is a major obstacle to successful chemotherapy in ovarian cancers. Identification and characterisation of chemoresponse-associated biomarkers are of paramount importance for novel therapeutic development. METHODS: Global RNA expression profiles were obtained by high-throughput microarray analysis. Cell cycle, proliferation rate, and paclitaxel sensitivity of ovarian cancer cells harbouring cyclin A1-inducible expression construct were compared with and without tetracycline induction, as well as when the cyclin A1 expression was suppressed by short inhibiting RNA (siRNA). Cellular senescence was evaluated by ß-galactosidase activity staining. RESULTS: Global RNA expression profiling and subsequent correlation studies of gene expression level and drug response has identified that elevated expression of cyclin A1 (CCNA1) was significantly associated with cellular resistance to paclitaxel, doxorubicin and 5-fluorouracil. The role of cyclin A1 in paclitaxel resistance was confirmed in ovarian cancer cells that harbour an inducible cyclin A1 expression construct, which showed reduced paclitaxel-mediated growth inhibition and apoptosis when cyclin A1 expression was induced, whereas downregulation of cyclin A1 expression in the same cell lines using cyclin A1-specific siRNAs sensitised the cells to paclitaxel toxicity. However, ovarian cancer cells with ectopic expression of cyclin A1 demonstrated slowdown of proliferation and senescence-associated ß-galactosidase activity. CONCLUSIONS: Our profiling and correlation studies have identified cyclin A1 as one chemoresistance-associated biomarker in ovarian cancer. The results of the characterisation studies suggest that cyclin A1 functions as an oncogene that controls proliferative and survival activities in tumourigenesis and chemoresistance of ovarian cancer.

Epithelialization of mouse ovarian tumor cells originating in the fallopian tube stroma.

Epithelial ovarian carcinoma accounts for 90% of all ovarian cancer and is the most deadly gynecologic malignancy. Recent studies have suggested that fallopian tube fimbriae can be the origin of cells for high-grade serous subtype of epithelial ovarian carcinoma (HGSOC). A mouse HGSOC model with conditional Dicer-Pten double knockout (Dicer-Pten DKO) developed primary tumors, intriguingly, from the fallopian tube stroma. We examined the growth and epithelial phenotypes of the Dicer-Pten DKO mouse tumor cells contributable by each gene knockout. Unlike human ovarian epithelial cancer cells that expressed full-length E-cadherin, the Dicer-Pten DKO stromal tumor cells expressed cleaved E-cadherin fragments and metalloproteinase 2, a mixture of epithelial and mesenchymal markers. Although the Dicer-Pten DKO tumor cells lost the expression of mature microRNAs as expected, they showed high levels of tRNA fragment expression and enhanced AKT activation due to the loss of PTEN function. Introduction of a Dicer1-expressing construct into the DKO mouse tumor cells significantly reduced DNA synthesis and the cell growth rate, with concurrent diminished adhesion and ZO1 epithelial staining. Hence, it is likely that the loss of Dicer promoted mesenchymal-epithelial transition in fallopian tube stromal cells, and in conjunction with Pten loss, further promoted cell proliferation and epithelial-like tumorigenesis.

Endosalpingiosis: More than just an incidental finding at the time of gynecologic surgery?

OBJECTIVE: To describe the clinical characteristics of patients with endosalpingiosis (ES) and examine its association with endometriosis and gynecologic malignancies. METHODS: We queried the medical record for patients who underwent gynecologic surgery (Gynecologic Surgery Cohort (GSC), n=58,161) from 1998 to 2013 at a single institution for the presence of "endosalpingiosis" (ES). Demographic and clinical characteristics were collected for patients with pathologically confirmed ES (n=838). Within GSC, we compared the frequency of endometriosis and gynecologic malignancies with and without ES. We estimated the expected distribution of ovarian cancer subtypes using cases from the New England Case Control Study (NECC). We used chi-square tests to test for significant differences in frequency distributions and unconditional logistic regression to calculate multivariate odds ratios for the association between ES and ovarian cancer subtypes. RESULTS: We observed concurrent endometriosis (p<0.0001), uterine cancer (p<0.0001), and ovarian cancer (p<0.0001) more frequently in women with ES. Women from the GSC with ES and ovarian cancer were more likely to have serous borderline (OR=10.2, 95% CI=5.1-20.7), clear cell (OR=3.0, 95% CI=1.1-8.0), and invasive mucinous tumors (OR=5.0, 95% CI=1.5-16.6) as compared to ovarian cancer cases from the NECC without ES, after accounting for age, race, menopausal status, parity, tubal ligation, and endometriosis. CONCLUSION: Women with ES are more likely to also be diagnosed with endometriosis, uterine, and ovarian cancers. Further study is needed to understand these associations so we may appropriately counsel patients with ES diagnosed at time of gynecologic surgery.

Pinin interacts with C-terminal binding proteins for RNA alternative splicing and epithelial cell identity of human ovarian cancer cells.

Unlike many other human solid tumors, ovarian tumors express many epithelial markers at a high level for cell growth and local invasion. The phosphoprotein Pinin plays a key role in epithelial cell identity. We showed that clinical ovarian tumors and ovarian cancer cell lines express a high level of Pinin when compared with normal ovarian tissues and immortalized normal ovarian surface epithelial cell lines. Pinin co-localized and physically interacted with transcriptional corepressor C-terminal binding proteins, CtBP1 and CtBP2, in the nuclei of cancer cells. Knockdown of Pinin in ovarian cancer cells resulted in specific reduction of CtBP1 protein expression, cell adhesion, anchorage-independent growth, and increased drug sensitivity. Whole transcriptomic comparison of next-generation RNA sequencing data between control ovarian cancer cell lines and cancer cell lines with respective knockdown of Pinin, CtBP1, and CtBP2 expression also showed reduced expression of CtBP1 mRNA in the Pinin knockdown cell lines. The Pinin knockdown cell lines shared significant overlap of differentially expressed genes and RNA splicing aberrations with CtBP1 knockdown and in a lesser degree with CtBP2 knockdown cancer cells. Hence, Pinin and CtBP are oncotargets that closely interact with each other to regulate transcription and pre-mRNA alternative splicing and promote cell adhesion and other epithelial characteristics of ovarian cancer cells.

Loss of E-cadherin disrupts ovarian epithelial inclusion cyst formation and collective cell movement in ovarian cancer cells.

Increased inclusion cyst formation in the ovary is associated with ovarian cancer development. We employed in vitro three-dimensional (3D) organotypic models formed by normal human ovarian surface epithelial (OSE) cells and ovarian cancer cells to study the morphologies of normal and cancerous ovarian cortical inclusion cysts and the molecular changes during their transitions into stromal microenvironment. When compared with normal cysts that expressed tenascin, the cancerous cysts expressed high levels of laminin V and demonstrated polarized structures in Matrigel; and the cancer cells migrated collectively when the cyst structures were positioned in a stromal-like collagen I matrix. The molecular markers identified in the in vitro 3D models were verified in clinical samples. Network analysis of gene expression of the 3D structures indicates concurrent downregulation of transforming growth factor beta pathway genes and high levels of E-cadherin and microRNA200 (miR200) expression in the cancerous cysts and the migrating cancer cells. Transient silencing of E-cadherin expression in ovarian cancer cells disrupted cyst structures and inhibited collective cell migration. Taken together, our studies employing 3D models have shown that E-cadherin is crucial for ovarian inclusion cyst formation and collective cancer cell migration.

Molecular changes in endometriosis-associated ovarian clear cell carcinoma.

BACKGROUND: Endometriosis is frequently associated with and thought of having propensity to develop into ovarian clear cell carcinoma (OCCC), although the molecular transformation mechanism is not completely understood. METHODS: We employed immunohistochemical (IHC) staining for marker expression along the potential progression continuum. Expression profiling of microdissected endometriotic and OCCC cells from patient-matched formalin-fixed, paraffin-embedded samples was performed to explore the carcinogenic pathways. Function of novel biomarkers was confirmed by knockdown experiments. RESULTS: PTEN was significantly lost in both endometriosis and invasive tumour tissues, while oestrogen receptor (ER) expression was lost in OCCC relative to endometriosis. XRCC5, PTCH2, eEF1A2 and PPP1R14B were significantly overexpressed in OCCC and associated endometriosis, but not in benign endometriosis (p ⩽ 0.004). Knockdown experiments with XRCC5 and PTCH2 in a clear cell cancer cell line resulted in significant growth inhibition. There was also significant silencing of a panel of target genes with histone H3 lysine 27 trimethylation, a signature of polycomb chromatin-remodelling complex in OCCC. IHC confirmed the loss of expression of one such polycomb target gene, the serous ovarian cancer lineage marker Wilms' tumour protein 1 (WT1) in OCCC, while endometriotic tissues showed significant co-expression of WT1 and ER. CONCLUSIONS: Loss of PTEN expression is proposed as an early and permissive event in endometriosis development, while the loss of ER and polycomb-mediated transcriptional reprogramming for pluripotency may play an important role in the ultimate transformation process. Our study provides new evidence to redefine the pathogenic programme for lineage-specific transformation of endometriosis to OCCC.

Inference on differences between classes using cluster-specific contrasts of mixed effects.

The detection of differentially expressed (DE) genes, that is, genes whose expression levels vary between two or more classes representing different experimental conditions (say, diseases), is one of the most commonly studied problems in bioinformatics. For example, the identification of DE genes between distinct disease phenotypes is an important first step in understanding and developing treatment drugs for the disease. We present a novel approach to the problem of detecting DE genes that is based on a test statistic formed as a weighted (normalized) cluster-specific contrast in the mixed effects of the mixture model used in the first instance to cluster the gene profiles into a manageable number of clusters. The key factor in the formation of our test statistic is the use of gene-specific mixed effects in the cluster-specific contrast. It thus means that the (soft) assignment of a given gene to a cluster is not crucial. This is because in addition to class differences between the (estimated) fixed effects terms for a cluster, gene-specific class differences also contribute to the cluster-specific contributions to the final form of the test statistic. The proposed test statistic can be used where the primary aim is to rank the genes in order of evidence against the null hypothesis of no DE. We also show how a P-value can be calculated for each gene for use in multiple hypothesis testing where the intent is to control the false discovery rate (FDR) at some desired level. With the use of publicly available and simulated datasets, we show that the proposed contrast-based approach outperforms other methods commonly used for the detection of DE genes both in a ranking context with lower proportion of false discoveries and in a multiple hypothesis testing context with higher power for a specified level of the FDR.

Tolerance induction to human stem cell transplants with extension to their differentiated progeny.

There is increasing interest in transplantation of human stem cells for therapeutic purposes. It would benefit future application if one could achieve their long-term acceptance and functional differentiation in allogeneic hosts using minimal immunosuppression. Allogeneic stem cell transplants differ from conventional tissue transplants insofar as not all alloantigens are revealed during tolerance induction. This risks that the immune system tolerized to antigens expressed by progenitors may still remain responsive to antigens expressed later during differentiation. Here we show that brief induction with monoclonal antibody-mediated coreceptor and costimulation blockade enables long-term engraftment and tolerance towards murine ESCs, hESCs, human induced pluripotent stem cells (iPSCs) and hESC-derived progenitors in outbred murine recipients. Tolerance induced to PSC-derived progenitors extends to their differentiated progenies, and sometimes even to different tissues derived from the same donor. Global gene expression profiling identifies clear features in T cells from tolerized grafts that are distinct from those involved in rejection.