The Salivary Mucin MG1 (MUC5B) Carries a Repertoire of Unique Oligosaccharides That is Large and Diverse

Glycobiology. Jan, 2002  |  Pubmed ID: 11825880

The high-molecular-mass salivary mucin MG1, one of two major mucins produced by human salivary glands, plays an important role in oral health by coating the tooth surface and by acting as a bacterial receptor. Here this mucin was purified from the submandibular/sublingual saliva of a blood group O individual. The presence of MUC5B as the major mucin in this preparation was confirmed by amino acid analysis and its reactivity with the monoclonal antibody PAN H2. To structurally characterize MG1 carbohydrates the O-glycans were released by reductive beta-elimination. Nuclear magnetic resonance spectroscopy of the nonfractionated mixture showed that (1) fucose was present in blood group H, Le(a), Le(x), Le(b), and Le(y) epitopes; (2) NeuAc was mainly linked alpha 2-3 to Gal or alpha 2-6 to GalNAcol; and (3) the major internal structures were core 1 and core 2 sequences. After this preliminary analysis the released oligosaccharides were separated into neutral (56%), sialylated (26%), and sulfated (19%) fractions, with an average length of 13, 17, and 41 sugar residues, respectively. Gas chromatography-mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of mixtures of neutral and sialylated oligosaccharides revealed at least 62 neutral and 25 sialylated oligosaccharides consisting of up to 20 monosaccharide residues. These results showed that the MG1-derived oligosaccharides were much longer than those of MG2, and only a few species were found on both molecules. Thus, these two mucins create an enormous repertoire of potential binding sites for microorganisms at one of the major portals where infectious organisms enter the body.

Vascular Endothelial Growth Factor Ligands and Receptors That Regulate Human Cytotrophoblast Survival Are Dysregulated in Severe Preeclampsia and Hemolysis, Elevated Liver Enzymes, and Low Platelets Syndrome

The American Journal of Pathology. Apr, 2002  |  Pubmed ID: 11943725

Human placental development combines elements of tumorigenesis and vasculogenesis. The organ's specialized epithelial cells, termed cytotrophoblasts, invade the uterus where they reside in the interstitial compartment. They also line uterine arteries and veins. During invasion, ectodermally derived cytotrophoblasts undergo pseudovasculogenesis, switching their adhesion molecule repertoire to mimic that of vascular cells. Failures in this transformation accompany the pregnancy complication preeclampsia. Here, we used a combination of in situ and in vitro analyses to characterize the cell's expression of vascular endothelial growth factor (VEGF) family ligands and receptors, key regulators of conventional vasculogenesis and angiogenesis. Cytotrophoblast differentiation and invasion during the first and second trimesters of pregnancy were associated with down-regulation of VEGF receptor (VEGFR)-2. Invasive cytotrophoblasts in early gestation expressed VEGF-A, VEGF-C, placental growth factor (PlGF), VEGFR-1, and VEGFR-3 and, at term, VEGF-A, PlGF, and VEGFR-1. In vitro the cells incorporated VEGF-A into the surrounding extracellular matrix; PlGF was secreted. We also found that cytotrophoblasts responded to the VEGF ligands they produced. Blocking ligand binding significantly decreased their expression of integrin alpha1, an adhesion molecule highly expressed by endovascular cytotrophoblasts, and increased apoptosis. In severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome, immunolocalization on tissue sections showed that cytotrophoblast VEGF-A and VEGFR-1 staining decreased; staining for PlGF was unaffected. Cytotrophoblast secretion of the soluble form of VEGFR-1 in vitro also increased. Together, the results of this study showed that VEGF family members regulate cytotrophoblast survival and that expression of a subset of family members is dysregulated in severe forms of preeclampsia.

Chemokine Expression and Function at the Human Maternal-fetal Interface

Reviews in Endocrine & Metabolic Disorders. May, 2002  |  Pubmed ID: 12007293

Trophoblast L-selectin-mediated Adhesion at the Maternal-fetal Interface

Science (New York, N.Y.). Jan, 2003  |  Pubmed ID: 12532021

Trophoblast adhesion to the uterine wall is the requisite first step of implantation and, subsequently, placentation. At the maternal-fetal interface, we investigated the expression of selectin adhesion systems that enable leukocyte capture from the bloodstream. On the maternal side, human uterine epithelial cells up-regulated selectin oligosaccharide-based ligands during the window of receptivity. On the fetal side, human trophoblasts expressed L-selectin. This ligand-receptor system was functional, because beads coated with the selectin ligand 6-sulfo sLe(x) bound to trophoblasts, and trophoblasts bound to ligand-expressing uterine luminal epithelium in tissue sections. These results suggest that trophoblast L-selectin mediates interactions with the uterus and that this adhesion mechanism may be critical to establishing human pregnancy.

Longitudinal Serum Concentrations of Placental Growth Factor: Evidence for Abnormal Placental Angiogenesis in Pathologic Pregnancies

American Journal of Obstetrics and Gynecology. Jan, 2003  |  Pubmed ID: 12548214

Complicated pregnancies demonstrate abnormal decidual and placental villous vasculature. We examined maternal concentrations of vascular endothelial growth factor and placental growth factor in normal pregnancies and in pregnancies that were complicated by isolated idiopathic small-for-gestational-age (SGA) newborn infants, preeclampsia alone, or preeclampsia with SGA newborn infants at the time of clinical disease and before the onset of clinical signs.

The Human Placenta Remodels the Uterus by Using a Combination of Molecules That Govern Vasculogenesis or Leukocyte Extravasation

Annals of the New York Academy of Sciences. May, 2003  |  Pubmed ID: 12814940

After fertilization, the next major hurdle for human reproduction is trophoblast differentiation, which is required for implantation, followed in lockstep by rapid assembly of these embryonic cells into a functional placenta. During this process, cytotrophoblast stem cells invade the uterus, anchoring the conceptus to the mother and establishing blood flow to the placenta. Cytotrophoblast invasion is actually a differentiation process that yields cells with many unusual attributes, that is, their tumor-like ability to invade the uterus and engraft maternal blood vessels. We discovered that once cytotrophoblasts commit to invasion they turn on expression of adhesion receptors characteristic of endothelium. They also begin to express and activate matrix metalloproteinase-9. Together, these phenotypic changes facilitate invasion while enabling cytotrophoblasts to present an endothelium-like surface to maternal blood. Currently, we are trying to understand the factors that play a role in cytotrophoblast differentiation/invasion. In keeping with the vascular characteristics that differentiated cytotrophoblasts assume, our recent data suggest that the key regulators include an unusual subset of vascular endothelial growth factor family members that play important roles in conventional vasculogenesis/angiogenesis. Surprisingly, we also discovered that these cells express functional l-selectin, which mediates neutrophil rolling and tethering, under shear stress, on inflamed endothelium. Trophoblast l-selectin likely interacts with carbohydrate selectin ligands that are upregulated on uterine glandular epithelium during the window of receptivity. Together, these data suggest that differentiating cytotrophoblasts have co-opted portions not only of vasculogenesis, but also of the process that facilitates leukocyte emigration from the blood into tissues, additional evidence of these cells' amazing plasticity.

A Positron-emitting Internal Marker for Identification of Normal Tissue by Positron Emission Tomography: Phantom Studies and Validation in Patients

Molecular Imaging and Biology : MIB : the Official Publication of the Academy of Molecular Imaging. Mar-Apr, 2003  |  Pubmed ID: 14499148

This study evaluated in a phantom model and verified in patients with lung cancer whether the use of an internal positron-emitting labeled marker could localize a critical structure by positron emission tomography (PET) imaging and verify multimodality image registration.

Disruption of Oxygen-regulated Responses Underlies Pathological Changes in the Placentas of Women Who Smoke or Who Are Passively Exposed to Smoke During Pregnancy

Reproductive Toxicology (Elmsford, N.Y.). Sep-Oct, 2003  |  Pubmed ID: 14555188

Previously, we showed that maternal smoking harms human placental development by changing the balance between cytotrophoblast (CTB) proliferation and differentiation. To understand the mechanisms involved, we studied the effects of maternal smoking and in vitro exposure of CTBs to nicotine and on CTB expression of molecules that govern cellular responses to oxygen tension: the von Hippel-Lindau tumor suppressor protein (pVHL), the hypoxia-inducible transcription factors (HIFs), and the vascular endothelial growth factors (VEGFs). We previously reported that hypoxia upregulates CTB pVHL expression (1). Here we show that in vitro exposure of CTBs to nicotine has the same effect. Maternal smoking also dysregulated CTB expression of all three molecules. Remarkably, we found that passive exposure to cigarette smoke had many of the same effects as active smoking, a graphic demonstration of the ill effects of cigarette smoke, even secondhand, on placental development. Together, these findings explain, in part, how smoking damages the placenta by altering expression of key mediators of placental development.

Human Cytotrophoblasts Promote Endothelial Survival and Vascular Remodeling Through Secretion of Ang2, PlGF, and VEGF-C

Developmental Biology. Nov, 2003  |  Pubmed ID: 14568550

Cytotrophoblasts are specialized epithelial cells of the human placenta that differentiate to acquire tumor-like properties that allow them to invade the uterus. Concurrently, they develop endothelial-like characteristics. This transformation allows cytotrophoblasts to replace the maternal cells that line uterine vessels, thereby diverting maternal blood to the placenta. Previously, we showed that invading cytotrophoblasts secrete VEGF-C and PlGF, factors that regulate their acquisition of an endothelial-like phenotype. Here, we examined the cells' expression of angiopoietin ligands and their Tie receptors. The data show that cytotrophoblasts predominantly expressed Ang2. We also studied the paracrine functions of Ang2 and the VEGFs by culturing uterine microvascular endothelial cells in cytotrophoblast-conditioned medium, which supported their growth. Removal of VEGF-C, PlGF, and/or Ang2 from the medium caused a marked reduction in cell number due to massive apoptosis. We also assayed the angiogenic potential of cytotrophoblast-derived factors in the chick chorioallantoic membrane assay. The results showed that they stimulated angiogenesis to a level comparable to that of basic FGF. These data suggest that invasive human cytotrophoblasts use an unusual repertoire of factors to influence the angiogenic state of maternal blood vessels and that this cross talk plays an important part in the endovascular component of uterine invasion.

Listeriosis in the Pregnant Guinea Pig: a Model of Vertical Transmission

Infection and Immunity. Jan, 2004  |  Pubmed ID: 14688130

Feto-placental infections represent a major cause of pregnancy complications, and yet the underlying molecular and cellular mechanisms of vertical transmission are poorly understood. Listeria monocytogenes, a facultative intracellular pathogen, is one of a group of pathogens that are known to cause feto-placental infections in humans and other mammals. The purpose of this study was to evaluate possible mechanisms of vertical transmission of L. monocytogenes. Humans and guinea pigs have a hemochorial placenta, where a single layer of fetally derived trophoblasts separates maternal from fetal circulation. We characterized L. monocytogenes infection of the feto-placental unit in a pregnant guinea pig model and in primary human trophoblasts and trophoblast-derived cell lines. The clinical manifestations of listeriosis in the pregnant guinea pigs and the tropism of L. monocytogenes to the guinea pig placenta resembled those in humans. Trophoblast cell culture systems were permissive for listerial growth and cell-to-cell spread and revealed that L. monocytogenes deficient in internalin A, a virulence factor that mediates invasion of nonphagocytic cells, was 100-fold defective in invasion. However, crossing of the feto-placental barrier in the guinea pig model was independent of internalin A, suggesting a negligible role for internalin-mediated direct invasion of trophoblasts in vivo. Further understanding of vertical transmission of L. monocytogenes will help in designing more effective means of treatment and disease prevention.

Reciprocal Chemokine Receptor and Ligand Expression in the Human Placenta: Implications for Cytotrophoblast Differentiation

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Apr, 2004  |  Pubmed ID: 15042711

At the onset of pregnancy, the human placenta, which forms the interface between the embryo/fetus and the mother, must rapidly develop into a life-sustaining organ. The many unusual processes entailed in placental development include the poorly understood phenomenon of maternal tolerance of the hemiallogeneic cells of the conceptus, including, most remarkably, placental trophoblasts that invade the uterine wall. To investigate whether this fetal organ exerts control over the maternal immune system at the level of leukocyte trafficking, we examined placental expression of chemokines, well-known cytokine regulators of leukocyte movements. In situ hybridization revealed abundant expression of 13 chemokines in the stromal but not the trophoblast compartment of chorionic villi. Potential roles for these molecules include recruitment of the resident macrophage (Hofbauer cell) population to the villi. In parallel, cytotrophoblast production of a panel of nine chemokine receptors was assessed by using RNase protection assays. The numerous receptors detected suggested the novel possibility that the paracrine actions of chemokine ligands derived from either the villous stroma or the decidua could mediate general aspects of placental development, with specific contributions to cytotrophoblast differentiation along the pathway that leads to uterine invasion.

Human Pregnancy: the Role of Chemokine Networks at the Fetal-maternal Interface

Expert Reviews in Molecular Medicine. May, 2004  |  Pubmed ID: 15130179

Chemokines are multifunctional molecules initially described as having a role in leukocyte trafficking and later found to participate in developmental processes such as differentiation and directed migration. Similar events occur in pregnancy during development of the fetal-maternal interface, where there is extensive leukocyte trafficking and tissue morphogenesis, and this is accompanied by abundant chemokine expression. The relationship between chemokines, leukocytes and placental development is beginning to be delineated. During pregnancy a specialised population of maternal leukocytes infiltrates the implantation site. These leukocytes are thought to sustain the delicate balance between protecting the developing embryo/fetus and tolerating its hemiallogeneic tissues. A network of chemokine expression by both fetal and maternal components in the pregnant uterus functions in establishing this leukocyte population. Intriguingly, experiments investigating immune cell recruitment revealed the additional possibility that chemokines influence aspects of placental development. Specifically, cytotrophoblasts, the effector cells of the placenta, express chemokine receptors that can bind ligands found at key locations, implicating chemokines as regulators of cytotrophoblast differentiation and migration. Thus, as in other systems, at the fetal-maternal interface chemokines might regulate multiple functions.

The Placental Problem: Linking Abnormal Cytotrophoblast Differentiation to the Maternal Symptoms of Preeclampsia

Reproductive Biology and Endocrinology : RB&E. Jul, 2004  |  Pubmed ID: 15236649

The placenta is a remarkable organ. In normal pregnancy its specialized cells (termed cytotrophoblasts) differentiate into various specialized subpopulations that play pivotal roles in governing fetal growth and development. One cytotrophoblast subset acquires tumor-like properties that allow the cells to invade the decidua and myometrium, a process that attaches the placenta to the uterus. The same subset also adopts a vascular phenotype that allows these fetal cells to breach and subsequently line uterine blood vessels, a process that channels maternal blood to the rest of the placenta. In the pregnancy complication preeclampsia, which is characterized by the sudden onset of maternal hypertension, proteinuria and edema, cytotrophoblast invasion is shallow and vascular transformation incomplete. These findings, together with very recent evidence from animal models, suggest that preeclampsia is associated with abnormal placental production of vasculogenic/angiogenic substances that reach the maternal circulation with the potential to produce at least a subset of the clinical signs of this syndrome. The current challenge is to build on this knowledge to design clinically useful tests for predicting, diagnosing and treating this dangerous disorder.

Trophoblast Differentiation During Embryo Implantation and Formation of the Maternal-fetal Interface

The Journal of Clinical Investigation. Sep, 2004  |  Pubmed ID: 15372095

Trophoblasts, the specialized cells of the placenta, play a major role in implantation and formation of the maternal-fetal interface. Through an unusual differentiation process examined in this review, these fetal cells acquire properties of leukocytes and endothelial cells that enable many of their specialized functions. In recent years a great deal has been learned about the regulatory mechanisms, from transcriptional networks to oxygen tension, which control trophoblast differentiation. The challenge is to turn this information into clinically useful tests for monitoring placental function and, hence, pregnancy outcome.

Trisomy 21 is Associated with Variable Defects in Cytotrophoblast Differentiation Along the Invasive Pathway

American Journal of Medical Genetics. Part A. Nov, 2004  |  Pubmed ID: 15389712

Aneuploid cells in the placenta are associated with poor pregnancy outcomes, but the mechanisms are unclear. Here, we examined the cytotrophoblast (CTB) differentiation pathway that leads to uterine invasion in pregnancies complicated by trisomy 21 (T21) as compared with their normal counterparts. Surprisingly, we observed a wide spectrum of T21 effects. Morphologically, some samples appeared near normal, while others had extensive fibrinoid deposition and apoptosis of CTBs at the maternal-fetal interface (confirmed by TUNEL labeling). At a molecular level, the cells' expression of stage-specific molecules was variably misregulated. At one end of the spectrum, samples with less apoptosis had relatively normal staining patterns. At the other end, samples with extensive apoptosis showed significantly decreased staining for these antigens. Additional studies confirmed that the effects we observed had functional consequences, because the cells exhibited marked phenotypic alterations in vitro, including a large increase in MMP-9 production, which distinguishes the effects of T21 on CTBs from those of preeclampsia. The morphologic, phenotypic, and functional differences among CTBs from pregnancies complicated by T21 illustrate the importance of the interplay between fetal/placental genotype and maternal influences on pregnancy outcome. Furthermore, our data may explain why a significant number of these pregnancies end in spontaneous abortion while others survive to term.

Abnormal Placentation and the Syndrome of Preeclampsia

Seminars in Nephrology. Nov, 2004  |  Pubmed ID: 15529288

Preeclampsia, particularly the severe cases that occur early in pregnancy, is associated with defects in the (placental) cytotrophoblast differentiation pathway that leads to uterine invasion. At a morphologic level, interstitial invasion often is shallow. Perhaps more significantly, endovascular invasion, particularly the arterial component, is rudimentary. The latter defect is thought to lead to hypoperfusion of the placenta. At a molecular level, these defects are associated with particular deficits in the differentiation process whereby cytotrophoblasts--epithelial cells of ectodermal origin--assume vascular-like properties. Until recently, the question was how the latter defects could lead to the maternal symptoms of this condition. Now a possible link in the form of preeclampsia-associated changes in placental production of vasculogenic/angiogenic substances and their inhibitors has been discovered. It is likely that this new paradigm will improve both diagnosis and treatment of this life-threatening pregnancy complication.

Highly Glycosylated Human Salivary Molecules Present Oligosaccharides That Mediate Adhesion of Leukocytes and Helicobacter Pylori

Biochemistry. Feb, 2005  |  Pubmed ID: 15697247

Glycoproteins display carbohydrate facets that serve as adhesion receptors for cells including leukocytes and bacterial cells. Our aim was to understand the role of the specialized carbohydrate motifs carried by highly glycosylated human salivary proteins in regulating the oral ecology. To date, our structural studies suggest that these molecules display a wide array of oligosaccharide structures, including many species with highly charged and/or fucosylated termini. Here, we used an immunoblot approach to gain additional information about the nature of these oligosaccharides. The results showed that MG1 and the salivary agglutinin express the MECA-79 epitope, an unusual sulfated carbohydrate structure that belongs to an important class of high-affinity (endothelial) L-selectin ligands. Unexpectedly, we discovered that in many women the expression of this epitope is hormonally regulated. Additional experiments revealed that MG1, MG2, and the salivary agglutinin also present Lewis blood group antigens, the exact repertoire varying on an individual basis. In parallel, we explored the functions of these carbohydrate motifs. Using an assay that detects L-selectin ligands, we found that the subset of MECA-79-reactive oligosaccharides displayed on salivary molecules specifically bind an L-selectin/Fc chimera. In contrast, the Lewis blood group structures are receptors for many strains of Helicobacter pylori, an organism that is implicated in the development of gastric ulcers and cancer. Together, these results suggest that MG1, MG2, and the salivary agglutinin play important roles in governing leukocyte and bacterial adhesion. Our findings suggest novel strategies, based on the relevant carbohydrate structures, for promoting or inhibiting these processes.

Toward Defining the Human Parotid Gland Salivary Proteome and Peptidome: Identification and Characterization Using 2D SDS-PAGE, Ultrafiltration, HPLC, and Mass Spectrometry

Biochemistry. Mar, 2005  |  Pubmed ID: 15723531

Saliva plays many biological roles, from lubrication and digestion to regulating bacterial and leukocyte adhesion. To understand the functions of individual components and families of molecules, it is important to identify as many salivary proteins as possible. Toward this goal, we used a proteomic approach as the first step in a global analysis of this important body fluid. We collected parotid saliva as the ductal secretion from three human donors and separated the protein components by two-dimensional SDS-polyacrylamide gel electrophoresis (2D SDS-PAGE). Proteins in gel spots were identified by peptide mass fingerprinting, and the results were confirmed by tandem mass spectrometry of selected peptides. Complementing this approach we used ultrafiltration to prepare a low-molecular-weight fraction of parotid saliva, which was analyzed directly or after reversed phase high-performance liquid chromatography separation by using mass spectrometric approaches. MS analyses of 2D SDS-PAGE spots revealed known components of saliva, including cystatins, histatins, lysozyme, and isoforms and/or fragments of alpha-amylase, albumin, and proline-rich proteins. We also discovered novel proteins, such as several isoforms of Zn-alpha-2-glycoprotein and secretory actin-binding protein. MS analyses of the ultrafiltrate showed that the low-molecular-weight fraction of parotid saliva was peptide-rich, with novel fragments of proline-rich proteins and histatins in abundance. Experiments using Candida albicans as the test organism showed that at least one of the novel peptides had antifungal activity. Our results show that saliva is a rich source of proteins and peptides that are potential diagnostic and therapeutic targets.

Human Cytotrophoblasts Acquire Aneuploidies As They Differentiate to an Invasive Phenotype

Developmental Biology. Mar, 2005  |  Pubmed ID: 15733669

Through an unusual differentiation process, human trophoblast progenitors (cytotrophoblasts) give rise to tumor-like cells that invade the uterus. By an unknown mechanism, invasive cytotrophoblasts exhibit permanent cell cycle withdrawal. Here, we report molecular cytogenetic data showing that approximately 20 to 60% of these interphase cells had acquired aneusomies involving chromosomes X, Y, or 16. The incidence positively correlated with gestational age and differentiation to an invasive phenotype. Scoring 12 chromosomes in flow-sorted cytotrophoblasts showed that more than 95% of the cells were hyperdiploid. Thus, aneuploidy appears to be an important component of normal placentation, perhaps limiting the proliferative and invasive potential of cytotrophoblasts within the uterus.

Serum-free Derivation of Human Embryonic Stem Cell Lines on Human Placental Fibroblast Feeders

Fertility and Sterility. May, 2005  |  Pubmed ID: 15866593

To derive new human embryonic stem cell (hESC) lines on pathogen-free human placental fibroblast feeders under serum-free conditions. Because the embryo develops in close contact with extraembryonic membranes, we hypothesized that placental mesenchyme might replicate the stem cell niche in situ.

Hypoxia-inducible Factor-dependent Histone Deacetylase Activity Determines Stem Cell Fate in the Placenta

Development (Cambridge, England). Aug, 2005  |  Pubmed ID: 15987772

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of HIFalpha and the arylhydrocarbon receptor nuclear translocator (ARNT/HIF1beta). Previously, we have reported that ARNT function is required for murine placental development. Here, we used cultured trophoblast stem (TS) cells to investigate the molecular basis of this requirement. In vitro, wild-type TS cell differentiation is largely restricted to spongiotrophoblasts and giant cells. Interestingly, Arnt-null TS cells differentiated into chorionic trophoblasts and syncytiotrophoblasts, as demonstrated by their expression of Tfeb, glial cells missing 1 (Gcm1) and the HIV receptor CXCR4. During this process, a region of the differentiating Arnt-null TS cells underwent granzyme B-mediated apoptosis, suggesting a role for this pathway in murine syncytiotrophoblast turnover. Surprisingly, HIF1alpha and HIF2alpha were induced during TS cell differentiation in 20% O2; additionally, pVHL levels were modulated during the same time period. These results suggest that oxygen-independent HIF functions are crucial to this differentiation process. As histone deacetylase (HDAC) activity has been linked to HIF-dependent gene expression, we investigated whether ARNT deficiency affects this epigenetic regulator. Interestingly, Arnt-null TS cells had reduced HDAC activity, increased global histone acetylation, and altered class II HDAC subcellular localization. In wild-type TS cells, inhibition of HDAC activity recapitulated the Arnt-null phenotype, suggesting that crosstalk between the HIFs and the HDACs is required for normal trophoblast differentiation. Thus, the HIFs play important roles in modulating the developmental plasticity of stem cells by integrating physiological, transcriptional and epigenetic inputs.

Assessing the Effects of Diurnal Variation on the Composition of Human Parotid Saliva: Quantitative Analysis of Native Peptides Using ITRAQ Reagents

Analytical Chemistry. Aug, 2005  |  Pubmed ID: 16053308

Changes in salivary composition correlate with disease susceptibility, disease state, or both. However, use of saliva for diagnostic purposes is complicated by the gland-specific effects of circadian rhythm or diurnal variation. We recently characterized a suite of peptides in the < or =10-kDa fraction of human parotid saliva that included many novel species. In this study, we used novel iTRAQ labeling chemistry to investigate possible diurnal effects on peptide generation. We collected samples produced by gustatory stimulation as the ductal secretions at four time points under conditions that minimized proteolysis, pooled them according to collection time, and isolated the LMW fractions. Samples collected at each collection time were derivatized with a different isobaric iTRAQ reagent. The labeled samples were combined, separated by reversed-phase HPLC, co-spotted with matrix on MALDI targets, and analyzed by MALDI TOF/TOF mass spectrometry. With this approach, we achieved relative quantification of the parotid peptides at four time points. In several cases, abundance during the day changed dramatically. iTRAQ tagging improved the efficiency of MS/MS fragmentation, which in turn allowed the identification of several novel peptides. Our results demonstrated both the utility of this method and the importance of diurnal effects on the composition of the human parotid saliva peptidome.

EPHB4 Regulates Chemokine-evoked Trophoblast Responses: a Mechanism for Incorporating the Human Placenta into the Maternal Circulation

Development (Cambridge, England). Sep, 2005  |  Pubmed ID: 16107476

In humans, fetal cytotrophoblasts leave the placenta and enter the uterine wall, where they preferentially remodel arterioles. The fundamental mechanisms that govern these processes are largely unknown. Previously, we have shown that invasive cytotrophoblasts express several chemokines, as well as the receptors with which they interact. Here, we report that these ligand-receptor interactions stimulate cytotrophoblast migration to approximately the same level as a growth factor cocktail that includes serum. Additionally, cytotrophoblast commitment to uterine invasion was accompanied by rapid downregulation of EPHB4, a transmembrane receptor associated with venous identity, and upregulation of ephrin B1. Within the uterine wall, the cells also upregulated expression of ephrin B2, an EPH transmembrane ligand that is associated with arterial identity. In vitro cytotrophoblasts avoided EPHB4-coated substrates; upon co-culture with 3T3 cells expressing this molecule, their migration was significantly inhibited. As to the mechanisms involved, cytotrophoblast interactions with EPHB4 downregulated chemokine-induced but not growth factor-stimulated migration. We propose that EPHB4/ephrin B1 interactions generate repulsive signals that direct cytotrophoblast invasion toward the uterus, where chemokines stimulate cytotrophoblast migration through the decidua. When cytotrophoblasts encounter EPHB4 expressed by venous endothelium, ephrin B-generated repulsive signals and a reduction in chemokine-mediated responses limit their interaction with veins. When they encounter ephrin B2 ligands expressed in uterine arterioles, migration is permitted. The net effect is preferential cytotrophoblast remodeling of arterioles, a hallmark of human placentation.

Functional and Placental Expression Analysis of the Human NRF3 Transcription Factor

Molecular Endocrinology (Baltimore, Md.). Jan, 2005  |  Pubmed ID: 15388789

Members of the Maf protooncogene and cap'n' collar families of basic-leucine zipper transcription factors play important roles in development, differentiation, oncogenesis, and stress signaling. In this study, we performed an in vivo protein-protein interaction screen to search for novel partners of the small Maf proteins. Using full-length human MAFG protein as bait, we identified the human basic-leucine zipper protein NRF3 [NF-E2 (nuclear factor erythroid 2)-related factor 3] as an interaction partner. Transfection studies confirmed that NRF3 is able to dimerize with MAFG. The resulting NRF3/MAFG heterodimer recognizes nuclear factor-erythroid 2/Maf recognition element-type DNA-binding motifs. Functional analysis revealed the presence of a strong transcriptional activation domain in the center region of the NRF3 protein. We found that NRF3 transcripts are present in placental chorionic villi from at least week 12 of gestation on through term. In particular, NRF3 is highly expressed in primary placental cytotrophoblasts, but not in placental fibroblasts. The human choriocarcinoma cell lines BeWo and JAR, derived from trophoblastic tumors of the placenta, also strongly express NRF3 transcripts. We generated a NRF3-specific antiserum and identified NRF3 protein in placental choriocarcinoma cells. Furthermore, we showed that NRF3 transcript and protein levels are induced by TNF-alpha in JAR cells. Our functional studies suggest that human NRF3 is a potent transcriptional activator. Finally, our expression and induction analyses hint at a possible role of Nrf3 in placental gene expression and development.

Modification of the Standard Trizol-based Technique Improves the Integrity of RNA Isolated from RNase-rich Placental Tissue

Clinical Chemistry. Jan, 2006  |  Pubmed ID: 16391340

Binding of the Streptococcal Surface Glycoproteins GspB and Hsa to Human Salivary Proteins

Infection and Immunity. Mar, 2006  |  Pubmed ID: 16495569

GspB and Hsa are homologous surface glycoproteins of Streptococcus gordonii that bind sialic acid moieties on platelet membrane glycoprotein Ibalpha. Since this species is an important member of the oral flora, we examined the direct binding of these adhesins to human salivary proteins. Both GspB and Hsa bound low-molecular-weight salivary mucin MG2 and salivary agglutinin. Hsa also bound several other salivary proteins, including secretory immunoglobulin A. Screening of six oral streptococcal isolates revealed that at least two of the strains expressed GspB homologues. These results indicate that GspB-like adhesins may be important for oral bacterial colonization.

MUC1 is a Scaffold for Selectin Ligands in the Human Uterus

Frontiers in Bioscience : a Journal and Virtual Library. 2006  |  Pubmed ID: 16720361

MUC1 is a large, transmembrane mucin glycoprotein abundantly expressed at the apical surface of uterine epithelia in all species examined to date. Loss of MUC1 at the time of embryo implantation occurs in many species; however, this does not appear to be the case in humans. Recent studies indicate that human blastocysts express L-selectin at their external surfaces raising the possibility that selectin ligands expressed at the apical surface of the uterine epithelium support early stages of blastocyst attachment. In the current study, we have used a panel of antibodies specific for selectin ligands to determine if MUC1 functions as a scaffold for these carbohydrate motifs in fertile women. The results demonstrate that MUC1 carries selectin ligands throughout the secretory phase of the menstrual cycle, including the mid-secretory (receptive) phase. Consequently, MUC1 represents a potential ligand for selectins expressed by human blastocysts.

Nicotine Downregulates the L-selectin System That Mediates Cytotrophoblast Emigration from Cell Columns and Attachment to the Uterine Wall

Reproductive Toxicology (Elmsford, N.Y.). Jul, 2006  |  Pubmed ID: 16806808

Here we show that maternal smoking downregulated, in a dose-dependent manner, cytotrophoblast expression of l-selectin and its TRA-1-81-reactive carbohydrate ligands. Cell islands -- cell columns that fail to make uterine attachments, often more numerous in the placentas of smokers -- exhibited an even greater downregulation of the l-selectin adhesion system. These effects were attributable to nicotine, since exposure of explanted villi to this drug in vitro reproduced the effects observed in situ. Videomicroscopy showed that the downstream consequences included inhibition of all stages of cytotrophoblast outgrowth from columns, including rolling adhesion within columns and generation of invasive cells at the distal ends. These results suggest that nicotine, acting through the l-selectin adhesion system, impairs the development of cell columns that connect the fetal portion of the placenta to the uterus, one possible reason why women who smoke have a much harder time achieving and sustaining pregnancy than their nonsmoking counterparts.

A Role for the L-selectin Adhesion System in Mediating Cytotrophoblast Emigration from the Placenta

Developmental Biology. Oct, 2006  |  Pubmed ID: 16930583

Cytotrophoblast (CTB) aggregates that bridge the gap between the placenta and the uterus are suspended as cell columns in the intervillous space, where they experience significant amounts of shear stress generated by maternal blood flow. The proper formation of these structures is crucial to pregnancy outcome as they play a vital role in anchoring the embryo/fetus to the decidua. At the same time, they provide a route by which CTBs enter the uterine wall. The mechanism by which the integrity of the columns is maintained while allowing cell movement is unknown. Here, we present evidence that the interactions of L-selectin with its carbohydrate ligands, a specialized adhesion system that is activated by shear stress, play an important role. CTBs in cell columns, particularly near the distal ends, stained brightly for L-selectin and with the TRA-1-81 antibody, which recognizes carbohydrate epitopes that support binding of L-selectin chimeras in vitro. Function-perturbing antibodies that inhibited either receptor or ligand activity also inhibited formation of cell columns in vitro. Together, these results suggest an autocrine role for the CTB L-selectin adhesion system in forming and maintaining cell columns during the early stages of placental development, when the architecture of the basal plate region is established. This type of adhesion may also facilitate CTB exit from cell columns, a prerequisite for uterine invasion.

Cytotrophoblast Induction of Arterial Apoptosis and Lymphangiogenesis in an in Vivo Model of Human Placentation

The Journal of Clinical Investigation. Oct, 2006  |  Pubmed ID: 16998586

We studied the vascular effects of invasive human cytotrophoblasts in vivo by transplanting placental villi to the fifth mammary fat pads or beneath the kidney capsules of Scid mice. Over 3 weeks, robust cytotrophoblast invasion was observed in both locations. The architecture of the mammary fat pad allowed for detailed analysis of the cells' interactions with resident murine blood vessels, which revealed specific induction of apoptosis in the endothelial cells and smooth muscle walls of the arterioles. This finding, and confirmation of the results in an in vitro coculture model, suggests that a parallel process is important for enabling cytotrophoblast endovascular invasion during human pregnancy. Cytotrophoblast invasion of the kidney parenchyma was accompanied by a robust lymphangiogenic response, while in vitro, the cells stimulated lymphatic endothelial cell migration via the actions of VEGF family members, FGF, and TNF-alpha. Immunolocalization analyses revealed that human pregnancy is associated with lymphangiogenesis in the decidua since lymphatic vessels were not a prominent feature of the nonpregnant endometrium. Thus, the placenta triggers the development of a decidual lymphatic circulation, which we theorize plays an important role in maintaining fluid balance during pregnancy, with possible implications for maternal-fetal immune cell trafficking.

Gene Expression Profiling of the Human Maternal-fetal Interface Reveals Dramatic Changes Between Midgestation and Term

Endocrinology. Mar, 2007  |  Pubmed ID: 17170095

Human placentation entails the remarkable integration of fetal and maternal cells into a single functional unit. In the basal plate region (the maternal-fetal interface) of the placenta, fetal cytotrophoblasts from the placenta invade the uterus and remodel the resident vasculature and avoid maternal immune rejection. Knowing the molecular bases for these unique cell-cell interactions is important for understanding how this specialized region functions during normal pregnancy with implications for tumor biology and transplantation immunology. Therefore, we undertook a global analysis of the gene expression profiles at the maternal-fetal interface. Basal plate biopsy specimens were obtained from 36 placentas (14-40 wk) at the conclusion of normal pregnancies. RNA was isolated, processed, and hybridized to HG-U133A&B Affymetrix GeneChips. Surprisingly, there was little change in gene expression during the 14- to 24-wk interval. In contrast, 418 genes were differentially expressed at term (37-40 wk) as compared with midgestation (14-24 wk). Subsequent analyses using quantitative PCR and immunolocalization approaches validated a portion of these results. Many of the differentially expressed genes are known in other contexts to be involved in differentiation, motility, transcription, immunity, angiogenesis, extracellular matrix dissolution, or lipid metabolism. One sixth were nonannotated or encoded hypothetical proteins. Modeling based on structural homology revealed potential functions for 31 of these proteins. These data provide a reference set for understanding the molecular components of the dialogue taking place between maternal and fetal cells in the basal plate as well as for future comparisons of alterations in this region that occur in obstetric complications.

Focal Adhesion Kinase Controls PH-dependent Epidermal Barrier Homeostasis by Regulating Actin-directed Na+/H+ Exchanger 1 Plasma Membrane Localization

The American Journal of Pathology. Jun, 2007  |  Pubmed ID: 17525272

Ubiquitously expressed focal adhesion kinase (FAK), linked to multiple intracellular signaling pathways, has previously been shown to control cell motility, invasion, proliferation, and survival. Using mice with a keratinocyte-restricted deletion of fak (FAK(K5 KO)), we report here a novel role for FAK: maintenance of adult epidermal permeability barrier homeostasis. Abundant lacunae of unprocessed lipids in stratum corneum (SC) of FAK(K5 KO) mice and delayed barrier recovery pointed to malfunction of pH-dependent enzymes active in extracellular space of SC. Measuring the SC pH gradient showed significantly more neutral pH values in FAK(K5 KO) mice, suggesting the importance of FAK for acidification. Moreover, normal functions were restored when FAK(K5 KO) mice were exposed to a surface pH typical of mouse SC (pH = 5.5). Baseline levels and response to barrier disruption of secretory phospholipase A2 isoforms, enzymes that mediate generation of free fatty acids in epidermis, appeared similar in both FAK(K5 KO) and control littermates. We found that the critical SC acidification regulator Na(+)/H(+) exchanger 1 failed to localize to the plasma membrane in FAK-deficient keratinocytes both in vivo and in vitro. Thus, for plasma membrane localization in terminally differentiated keratinocytes, Na(+)/H(+) exchanger 1 requires an intact actin cytoskeleton, which is impaired in FAK-deficient cells.

Comparative Analysis of Maternal-fetal Interface in Preeclampsia and Preterm Labor

Cell and Tissue Research. Sep, 2007  |  Pubmed ID: 17549520

The maternal-fetal interface, a chimeric structure, is formed when fetal cytotrophoblasts (CTBs) from the placenta invade the uterine wall and its resident vasculature. In preeclampsia (PE), interstitial and endovascular invasion are often shallow, and fewer spiral arterioles are breached in toto. Our previous work has shown that faulty CTB differentiation to an invasive phenotype is a contributing factor. Here, we have tested the hypothesis that the constellation of morphological and molecular defects that are associated with PE are unique to this condition. Specifically, we have compared the histology of the maternal-fetal interface and CTB expression of stage-specific antigens in PE and in preterm labor (PTL) with or without inflammation. In the absence of inflammation, biopsies obtained after PTL were near normal at histological and molecular levels. In accord with previously published data, PE had severe negative effects on the endpoints analyzed. Biopsies obtained after PTL with inflammation had an intermediate phenotype. Our results suggest that the maternal-fetal interface from cases of PTL without inflammation can be used for comparative purposes, e.g., as age-matched controls, in studies of the effects of PE on cells in this region.

Disruption of Apical-basal Polarity of Human Embryonic Stem Cells Enhances Hematoendothelial Differentiation

Stem Cells (Dayton, Ohio). Sep, 2007  |  Pubmed ID: 17569786

During murine development, the formation of tight junctions and acquisition of polarity are associated with allocation of the blastomeres on the outer surface of the embryo to the trophoblast lineage, whereas the absence of polarization directs cells to the inner cell mass. Here, we report the results of ultrastructural analyses that suggest a similar link between polarization and cell fate in human embryos. In contrast, the five human embryonic stem cell (hESC) lines displayed apical-basal, epithelial-type polarity with electron-dense tight junctions, apical microvilli, and asymmetric distribution of organelles. Consistent with these findings, molecules that are components of tight junctions or play regulatory roles in polarization localized to the apical regions of the hESCs at sites of cell-cell contact. The tight junctions were functional, as shown by the ability of hESC colonies to exclude the pericellular passage of a biotin compound. Depolarization of hESCs produced multilayered aggregates of rapidly proliferating cells that continued to express transcription factors that are required for pluripotency at the same level as control cells. However, during embryoid body formation, depolarized cells differentiated predominantly along mesenchymal lineage and spontaneously produced hematoendothelial precursors more efficiently than control ESC. Our findings have numerous implications with regard to strategies for deriving, propagating, and differentiating hESC.

A Mass Spectrometry-based Strategy for Detecting and Characterizing Endogenous Proteinase Activities in Complex Biological Samples

Proteomics. Feb, 2008  |  Pubmed ID: 18186022

Endogenous proteinases in biological fluids such as human saliva produce a rich peptide repertoire that reflects a unique combination of enzymes, substrates, and inhibitors/activators. Accordingly, this subproteome is an interesting source of biomarkers for disease processes that either directly or indirectly involve proteolysis. However, the relevant proteinases, typically very low abundance molecules, are difficult to classify and identify. We hypothesized that a sensitive technique for monitoring accumulated peptide products in an unbiased, global manner would be very useful for detecting and profiling proteolytic activities in complex biological samples. Building on the longstanding use of 18O isotope-based approaches for the classification of proteolytic and other enzymatic processes we devised a new method for evaluating endogenous proteinases. Specifically, we showed that upon ex vivo incubation endogenous proteinases in human parotid saliva introduced 18O from isotopically enriched water into the C-terminal carboxylic groups of their peptide products. Subsequent peptide sequence determination and inhibitor profiling enabled the detection of discrete subsets of proteolytic products that were generated by different enzymes. As a proof-of-principle we used one of these fingerprints to identify the relevant activity as tissue kallikrein. We termed this technique PALeO. Our results suggest that PALeO is a rapid and highly sensitive method for globally assessing proteinase activities in complex biological samples.

Nuclear FAK Promotes Cell Proliferation and Survival Through FERM-enhanced P53 Degradation

Molecular Cell. Jan, 2008  |  Pubmed ID: 18206965

FAK is known as an integrin- and growth factor-associated tyrosine kinase promoting cell motility. Here we show that, during mouse development, FAK inactivation results in p53- and p21-dependent mesodermal cell growth arrest. Reconstitution of primary FAK-/-p21-/- fibroblasts revealed that FAK, in a kinase-independent manner, facilitates p53 turnover via enhanced Mdm2-dependent p53 ubiquitination. p53 inactivation by FAK required FAK FERM F1 lobe binding to p53, FERM F2 lobe-mediated nuclear localization, and FERM F3 lobe for connections to Mdm2 and proteasomal degradation. Staurosporine or loss of cell adhesion enhanced FERM-dependent FAK nuclear accumulation. In primary human cells, FAK knockdown raised p53-p21 levels and slowed cell proliferation but did not cause apoptosis. Notably, FAK knockdown plus cisplatin triggered p53-dependent cell apoptosis, which was rescued by either full-length FAK or FAK FERM re-expression. These studies define a scaffolding role for nuclear FAK in facilitating cell survival through enhanced p53 degradation under conditions of cellular stress.

A "tagless" Strategy for Identification of Stable Protein Complexes Genome-wide by Multidimensional Orthogonal Chromatographic Separation and ITRAQ Reagent Tracking

Journal of Proteome Research. May, 2008  |  Pubmed ID: 18336004

Tandem affinity purification is the principal method for purifying and identifying stable protein complexes system-wide in whole cells. Although highly effective, this approach is laborious and impractical in organisms where genetic manipulation is not possible. Here, we propose a novel "tagless" strategy that combines multidimensional separation of endogenous complexes with mass spectrometric monitoring of their composition. In this procedure, putative protein complexes are identified based on the comigration of collections of polypeptides through multiple orthogonal separation steps. We present proof-of-principle evidence for the feasibility of key aspects of this strategy. A majority of Escherichia coli proteins are shown to remain in stable complexes during fractionation of a crude extract through three chromatographic steps. We also demonstrate that iTRAQ reagent-based tracking can quantify relative migration of polypeptides through chromatographic separation media. LC MALDI MS and MS/MS analysis of the iTRAQ-labeled peptides gave reliable relative quantification of 37 components of 13 known E. coli complexes: 95% of known complex components closely co-eluted and 57% were automatically grouped by a prototype computational clustering method. With further technological improvements in each step, we believe this strategy will dramatically improve the efficiency of the purification and identification of protein complexes in cells.

The Proteomes of Human Parotid and Submandibular/sublingual Gland Salivas Collected As the Ductal Secretions

Journal of Proteome Research. May, 2008  |  Pubmed ID: 18361515

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

Chapter 12. Placental Remodeling of the Uterine Vasculature

Methods in Enzymology. 2008  |  Pubmed ID: 19022064

In eutherian mammals, the first functional organ is the placenta, a transient structure that is rapidly assembled in the extraembryonic compartment. By necessity the placenta develops in advance of the embryo, which it supports in utero by performing many of the same functions that the lungs, gastrointestinal tract, and urinary system carry out after birth. Specialized epithelial cells that arise from the placenta, termed cytotrophoblasts (CTBs), are responsible for redirecting maternal blood to the developing conceptus, which occurs as a result of the cells' aggressive invasion through the maternal endometrial stroma (interstitial invasion) and resident blood vessels (endovascular invasion). The latter process involves displacement of maternal endothelium and induction of apoptosis in the surrounding smooth muscle. Together, these events result in a reduction of blood vessel elasticity and increased blood flow. In the past, investigations of human CTB endovascular invasion have been limited to immunohistochemical examination of tissue sections. In this chapter, we will discuss the use of in vitro and in vivo techniques that have been recently adapted for the study of the complex events that occur during CTB endovascular invasion. As an introduction, we provide background on placental anatomy and the molecular basis of CTB behaviors. To follow, we present techniques used in the isolation and culture of primary CTBs and chorionic villous explants. Approaches for identifying trophoblast-modified blood vessels in placental tissue sections are also described. Next, we review methods used by other groups to study CTB/endothelial interactions in culture focusing on techniques that employ isolated cells and chorionic explants. Finally, we conclude with methods devised by our group and others to explore the complex heterotypic cell-cell interactions that occur as CTBs invade blood vessels in vivo in the nude mouse.

The Human Placenta is a Hematopoietic Organ During the Embryonic and Fetal Periods of Development

Developmental Biology. Mar, 2009  |  Pubmed ID: 19073167

We studied the potential role of the human placenta as a hematopoietic organ during embryonic and fetal development. Placental samples contained two cell populations-CD34(++)CD45(low) and CD34(+)CD45(low)-that were found in chorionic villi and in the chorioamniotic membrane. CD34(++)CD45(low) cells express many cell surface antigens found on multipotent primitive hematopoietic progenitors and hematopoietic stem cells. CD34(++)CD45(low) cells contained colony-forming units culture (CFU-C) with myeloid and erythroid potential in clonogenic in vitro assays, and they generated CD56(+) natural killer cells and CD19(+)CD20(+)sIgM(+) B cells in polyclonal liquid cultures. CD34(+)CD45(low) cells mostly comprised erythroid- and myeloid-committed progenitors, while CD34(-) cells lacked CFU-C. The placenta-derived precursors were fetal in origin, as demonstrated by FISH using repeat-sequence chromosome-specific probes for X and Y. The number of CD34(++)CD45(low) cells increased with gestational age, but their density (cells per gram of tissue) peaked at 5-8 wk, decreasing more than sevenfold at the onset of the fetal phase (9 wk of gestation). In addition to multipotent progenitors, the placenta contained myeloid- and erythroid-committed progenitors indicative of active in situ hematopoiesis. These data suggest that the human placenta is an important hematopoietic organ, raising the possibility of banking placental hematopoietic stem cells along with cord blood for transplantation.

A New Role for the Human Placenta As a Hematopoietic Site Throughout Gestation

Reproductive Sciences (Thousand Oaks, Calif.). Feb, 2009  |  Pubmed ID: 19208786

We investigated whether the human placenta contributes to embryonic and fetal hematopoietic development. Two cell populations--CD34(++)CD45(low) and CD34( +)CD45(low)--were found in chorionic villi. CD34(++) CD45(low) cells display many markers that are characteristic of multipotent primitive hematopoietic progenitors and hematopoietic stem cells. Clonogenic in vitro assays showed that CD34(++)CD45( low) cells contained colony-forming units-culture with myeloid and erythroid potential and differentiated into CD56(+) natural killer cells and CD19(+) B cells in culture. CD34(+)CD45(low) cells were mostly enriched in erythroid- and myeloid-committed progenitors. While the number of CD34(++)CD45(low) cells increased throughout gestation in parallel with placental mass. However, their density (cells per gram of tissue) reached its peak at 5 to 8 weeks, decreasing more than 7-fold from the ninth week onward. In addition to multipotent progenitors, the placenta contained intermediate progenitors, indicative of active hematopoiesis. Together, these data suggest that the human placenta is potentially an important hematopoietic organ, opening the possibility of banking placental hematopoietic stem cells along with cord blood for transplantation.

Derivation of Human Embryonic Stem Cell Lines from Biopsied Blastomeres on Human Feeders with Minimal Exposure to Xenomaterials

Stem Cells and Development. Nov, 2009  |  Pubmed ID: 19222349

In a continuous effort to improve the generation of therapeutic grade human embryonic stem cell (hESC) lines, we focused on preserving developmental capacity of the embryos, minimizing the exposure to xenomaterials, increasing derivation efficacy, and reducing the complexity of the derivation procedure. In this study, we describe an improved method for efficient derivation of hESC lines from blastomeres of biopsied embryos. Our protocol substituted feeder cells of mouse origin with human foreskin fibroblasts (HFFs), limited serum exposure of cells to formation of the initial outgrowth, and increased derivation efficacy from 12.5% (one hESC line out of 13 biopsies) to 50% (3 out of 6 biopsies) by using early population doubling (PD) HFFs. In addition, it eliminated a need for embryo-blastomere coculture, thus reducing the complexity of the culture and enabling continued development of the biopsied embryo under optimal conditions. All derived lines maintained normal karyotype and expressed totipotent phenotype including the ability to differentiate into trophectoderm and all three germ layers.

Acid-catalyzed Oxygen-18 Labeling of Peptides

Analytical Chemistry. Apr, 2009  |  Pubmed ID: 19243188

In enzymatic (18)O-labeling strategies for quantitative proteomics, the exchange of carboxyl oxygens at low pH is a common, undesired side reaction. We asked if acid-catalyzed back exchange could interfere with quantitation and whether the reaction itself could be used as method for introducing (18)O label into peptides. Several synthetic peptides were dissolved in dilute acid containing 50% (v/v) H(2)(18)O and incubated at room temperature. Aliquots were removed over a period of 3 weeks and analyzed by tandem mass spectrometry (MS/MS). (18)O-incorporation ratios were determined by linear regression analysis that allowed for multiple stable-isotope incorporations. At low pH, peptides exchanged their carboxyl oxygen atoms with the aqueous solvent. The isotope patterns gradually shifted to higher masses until they reached the expected binomial distribution at equilibrium after approximately 11 days. Reaction rates were residue- and sequence-specific. Due to its slow nature, the acid-catalyzed back exchange is expected to minimally interfere with enzymatic (18)O-labeling studies provided that storage and analysis conditions minimize low-pH exposure times. On its own, acid-catalyzed (18)O labeling is a general tagging strategy that is an alternative to the chemical, metabolic, and enzymatic isotope-labeling schemes currently used in quantitative proteomics.

MTOR Supports Long-term Self-renewal and Suppresses Mesoderm and Endoderm Activities of Human Embryonic Stem Cells

Proceedings of the National Academy of Sciences of the United States of America. May, 2009  |  Pubmed ID: 19416884

Despite the recent identification of the transcriptional regulatory circuitry involving SOX2, NANOG, and OCT-4, the intracellular signaling networks that control pluripotency of human embryonic stem cells (hESCs) remain largely undefined. Here, we demonstrate an essential role for the serine/threonine protein kinase mammalian target of rapamycin (mTOR) in regulating hESC long-term undifferentiated growth. Inhibition of mTOR impairs pluripotency, prevents cell proliferation, and enhances mesoderm and endoderm activities in hESCs. At the molecular level, mTOR integrates signals from extrinsic pluripotency-supporting factors and represses the transcriptional activities of a subset of developmental and growth-inhibitory genes, as revealed by genome-wide microarray analyses. Repression of the developmental genes by mTOR is necessary for the maintenance of hESC pluripotency. These results uncover a novel signaling mechanism by which mTOR controls fate decisions in hESCs. Our findings may contribute to effective strategies for tissue repair and regeneration.

Multi-site Assessment of the Precision and Reproducibility of Multiple Reaction Monitoring-based Measurements of Proteins in Plasma

Nature Biotechnology. Jul, 2009  |  Pubmed ID: 19561596

Verification of candidate biomarkers relies upon specific, quantitative assays optimized for selective detection of target proteins, and is increasingly viewed as a critical step in the discovery pipeline that bridges unbiased biomarker discovery to preclinical validation. Although individual laboratories have demonstrated that multiple reaction monitoring (MRM) coupled with isotope dilution mass spectrometry can quantify candidate protein biomarkers in plasma, reproducibility and transferability of these assays between laboratories have not been demonstrated. We describe a multilaboratory study to assess reproducibility, recovery, linear dynamic range and limits of detection and quantification of multiplexed, MRM-based assays, conducted by NCI-CPTAC. Using common materials and standardized protocols, we demonstrate that these assays can be highly reproducible within and across laboratories and instrument platforms, and are sensitive to low mug/ml protein concentrations in unfractionated plasma. We provide data and benchmarks against which individual laboratories can compare their performance and evaluate new technologies for biomarker verification in plasma.

Severe Preeclampsia-related Changes in Gene Expression at the Maternal-fetal Interface Include Sialic Acid-binding Immunoglobulin-like Lectin-6 and Pappalysin-2

Endocrinology. Jan, 2009  |  Pubmed ID: 18818296

Preeclampsia (PE), which affects 4-8% of human pregnancies, causes significant maternal and neonatal morbidity and mortality. Within the basal plate, placental cytotrophoblasts (CTBs) of fetal origin invade the uterus and extensively remodel the maternal vasculature. In PE, CTB invasion is often shallow, and vascular remodeling is rudimentary. To better understand possible causes, we conducted a global analysis of gene expression at the maternal-fetal interface in placental samples from women with PE (n = 12; 24-36 wk) vs. samples from women who delivered due to preterm labor with no evidence of infection (n = 11; 24-36 wk), a condition that our previous work showed is associated with normal CTB invasion. Using the HG-U133A&B Affymetrix GeneChip platform, and statistical significance set at log odds-ratio of B >0, 55 genes were differentially expressed in PE. They encoded proteins previously associated with PE [e.g. Flt-1 (vascular endothelial growth factor receptor-1), leptin, CRH, and inhibin] and novel molecules [e.g. sialic acid binding Ig-like lectin 6 (Siglec-6), a potential leptin receptor, and pappalysin-2 (PAPP-A2), a protease that cleaves IGF-binding proteins]. We used quantitative PCR to validate the expression patterns of a subset of the genes. At the protein level, we confirmed PE-related changes in the expression of Siglec-6 and PAPP-A2, which localized to invasive CTBs and syncytiotrophoblasts. Notably, Siglec-6 placental expression is uniquely human, as is spontaneous PE. The functional significance of these novel observations may provide new insights into the pathogenesis of PE, and assaying the circulating levels of these proteins could have clinical utility for predicting and/or diagnosing PE.

Interlaboratory Study Characterizing a Yeast Performance Standard for Benchmarking LC-MS Platform Performance

Molecular & Cellular Proteomics : MCP. Feb, 2010  |  Pubmed ID: 19858499

Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LC-MS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories; the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize pre-analytical and analytical variation in comparative proteomics experiments.

Repeatability and Reproducibility in Proteomic Identifications by Liquid Chromatography-tandem Mass Spectrometry

Journal of Proteome Research. Feb, 2010  |  Pubmed ID: 19921851

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.

Protein-based Multiplex Assays: Mock Presubmissions to the US Food and Drug Administration

Clinical Chemistry. Feb, 2010  |  Pubmed ID: 20007858

As a part of ongoing efforts of the NCI-FDA Interagency Oncology Task Force subcommittee on molecular diagnostics, members of the Clinical Proteomic Technology Assessment for Cancer program of the National Cancer Institute have submitted 2 protein-based multiplex assay descriptions to the Office of In Vitro Diagnostic Device Evaluation and Safety, US Food and Drug Administration. The objective was to evaluate the analytical measurement criteria and studies needed to validate protein-based multiplex assays. Each submission described a different protein-based platform: a multiplex immunoaffinity mass spectrometry platform for protein quantification, and an immunological array platform quantifying glycoprotein isoforms. Submissions provided a mutually beneficial way for members of the proteomics and regulatory communities to identify the analytical issues that the field should address when developing protein-based multiplex clinical assays.

Analytical Validation of Protein-based Multiplex Assays: a Workshop Report by the NCI-FDA Interagency Oncology Task Force on Molecular Diagnostics

Clinical Chemistry. Feb, 2010  |  Pubmed ID: 20007859

Clinical proteomics has the potential to enable the early detection of cancer through the development of multiplex assays that can inform clinical decisions. However, there has been some uncertainty among translational researchers and developers as to the specific analytical measurement criteria needed to validate protein-based multiplex assays. To begin to address the causes of this uncertainty, a day-long workshop titled "Interagency Oncology Task Force Molecular Diagnostics Workshop" was held in which members of the proteomics and regulatory communities discussed many of the analytical evaluation issues that the field should address in development of protein-based multiplex assays for clinical use. This meeting report explores the issues raised at the workshop and details the recommendations that came out of the day's discussions, such as a workshop summary discussing the analytical evaluation issues that specific proteomic technologies should address when seeking US Food and Drug Administration approval.

The Placenta: Transcriptional, Epigenetic, and Physiological Integration During Development

The Journal of Clinical Investigation. Apr, 2010  |  Pubmed ID: 20364099

The placenta provides critical transport functions between the maternal and fetal circulations during intrauterine development. Formation of this interface relies on coordinated interactions among transcriptional, epigenetic, and environmental factors. Here we describe these mechanisms in the context of the differentiation of placental cells (trophoblasts) and synthesize current knowledge about how they interact to generate a functional placenta. Developing an understanding of these pathways contributes to an improvement of our models for studying trophoblast biology and sheds light on the etiology of pregnancy complications and the in utero programming of adult diseases.

Performance Metrics for Liquid Chromatography-tandem Mass Spectrometry Systems in Proteomics Analyses

Molecular & Cellular Proteomics : MCP. Feb, 2010  |  Pubmed ID: 19837981

A major unmet need in LC-MS/MS-based proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LC-MS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations. The metrics typically displayed variations less than 10% and thus can reveal even subtle differences in performance of system components. Analyses of data from interlaboratory studies conducted under a common standard operating procedure identified outlier data and provided clues to specific causes. Moreover, interlaboratory variation reflected by the metrics indicates which system components vary the most between laboratories. Application of these metrics enables rational, quantitative quality assessment for proteomics and other LC-MS/MS analytical applications.

Sweetening the Pot: Adding Glycosylation to the Biomarker Discovery Equation

Clinical Chemistry. Feb, 2010  |  Pubmed ID: 19959616

Cancer has profound effects on gene expression, including a cell's glycosylation machinery. Thus, tumors produce glycoproteins that carry oligosaccharides with structures that are markedly different from the same protein produced by a normal cell. A single protein can have many glycosylation sites that greatly amplify the signals they generate compared with their protein backbones.

A Lectin Affinity Workflow Targeting Glycosite-specific, Cancer-related Carbohydrate Structures in Trypsin-digested Human Plasma

Analytical Biochemistry. Jan, 2011  |  Pubmed ID: 20705048

Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.

Trophoblast Stem Cells

Biology of Reproduction. Mar, 2011  |  Pubmed ID: 21106963

Trophoblast stem cells (TSC) are the precursors of the differentiated cells of the placenta. In the mouse, TSC can be derived from outgrowths of either blastocyst polar trophectoderm (TE) or extraembryonic ectoderm (ExE), which originates from polar TE after implantation. The mouse TSC niche appears to be located within the ExE adjacent to the epiblast, on which it depends for essential growth factors, but whether this cellular architecture is the same in other species remains to be determined. Mouse TSC self-renewal can be sustained by culture on mitotically inactivated feeder cells, which provide one or more factors related to the NODAL pathway, and a medium supplemented with FGF4, heparin, and fetal bovine serum. Repression of the gene network that maintains pluripotency and emergence of the transcription factor pathways that specify a trophoblast (TR) fate enables TSC derivation in vitro and placental formation in vivo. Disrupting the pluripotent network of embryonic stem cells (ESC) causes them to default to a TR ground state. Pluripotent cells that have acquired sublethal chromosomal alterations may be sequestered into TR for similar reasons. The transition from ESC to TSC, which appears to be unidirectional, reveals important aspects of initial fate decisions in mice. TSC have yet to be derived from domestic species in which remarkable TR growth precedes embryogenesis. Recent derivation of TSC from blastocysts of the rhesus monkey suggests that isolation of the human equivalents may be possible and will reveal the extent to which mechanisms uncovered by using animal models are true in our own species.

Transcriptomic Signature of Trophoblast Differentiation in a Human Embryonic Stem Cell Model

Biology of Reproduction. Jun, 2011  |  Pubmed ID: 21368299

Identification of genes involved in trophoblast differentiation is of great interest in understanding cellular and molecular mechanisms involved in placental development and is relevant clinically to fetal development, fertility, and maternal health. Herein, we investigated differentiation of human embryonic stem cells (hESCs) down the trophoblast lineage by culture with bone morphogenetic protein 4 (BMP4) over a 10-day period. Within 2 days, the stemness markers POU5F1 and NANOG were markedly down-regulated, followed temporally by up-regulation of the CDX2, KRT7, HLA-G, ID2, CGA, and CGB trophoblast markers. To understand, on a global scale, changes in the transcriptome during the differentiation of hESCs down the trophoblast lineage, a large-scale microarray analysis was performed. Through whole-genome analysis, more than 3800 genes displayed statistically significant and 2-fold or greater changes in expression during the time course. Of those genes that showed the largest increases, many were involved in processes associated with trophoblast biology; however, novel genes were also identified. Some of them are hypothesized to be associated mainly with extracellular matrix remodeling (e.g., NID2) and cell migration and invasion (e.g., RAB25). Using Ingenuity pathways analysis software to identify signaling pathways involved in trophoblast differentiation or function, we discovered that many genes are involved in WNT/beta-catenin, ERK/MAPK, NFKB, and calcium signaling pathways, suggesting potential roles for these families in trophoblast development. This work provides an in vitro functional genomic model with which to identify genes involved in trophoblast development.

SGK1: a Fine Balancing Act for Human Pregnancy

Nature Medicine. 2011  |  Pubmed ID: 22064410

Human Placenta and Chorion: Potential Additional Sources of Hematopoietic Stem Cells for Transplantation

Transfusion. Nov, 2011  |  Pubmed ID: 22074633

Hematopoietic stem cell (HSC) transplantation is an essential element of medical therapy, leading to cures of previously incurable hematological and nonhematological diseases. Many patients do not find matched donors in a timely manner, which has driven efforts to find alternative pools of transplantable HSCs. The use of umbilical cord blood (UCB) as a source of transplantable HSCs began more than two decades ago. However, the use of UCB as a reliable source of HSCs for transplantation still faces crucial challenges: the number of HSCs present in a unit of UCB is usually sufficient for younger children but not for adults, and the persistent delayed engraftment often seen can result in high rates of infection and mortality.

GROα Regulates Human Embryonic Stem Cell Self-renewal or Adoption of a Neuronal Fate

Differentiation; Research in Biological Diversity. Apr, 2011  |  Pubmed ID: 21396766

Previously we reported that feeders formed from human placental fibroblasts (hPFs) support derivation and long-term self-renewal of human embryonic stem cells (hESCs) under serum-free conditions. Here, we show, using antibody array and ELISA platforms, that hPFs secrete ∼6-fold higher amounts of the CXC-type chemokine, GROα, than IMR 90, a human lung fibroblast line, which does not support hESC growth. Furthermore, immunocytochemistry and immunoblot approaches revealed that hESCs express CXCR, a GROα receptor. We used this information to develop defined culture medium for feeder-free propagation of hESCs in an undifferentiated state. Cells passaged as small aggregates and maintained in the GROα-containing medium had a normal karyotype, expressed pluripotency markers, and exhibited apical-basal polarity, i.e., had the defining features of pluripotent hESCs. They also differentiated into the three primary (embryonic) germ layers and formed teratomas in immunocompromised mice. hESCs cultured as single cells in the GROα-containing medium also had a normal karyotype, but they downregulated markers of pluripotency, lost apical-basal polarity, and expressed markers that are indicative of the early stages of neuronal differentiation-βIII tubulin, vimentin, radial glial protein, and nestin. These data support our hypothesis that establishing and maintaining cell polarity is essential for the long-term propagation of hESCs in an undifferentiated state and that disruption of cell-cell contacts can trigger adoption of a neuronal fate.

Pre-eclampsia is Associated with Elevated CXCL12 Levels in Placental Syncytiotrophoblasts and Maternal Blood

European Journal of Obstetrics, Gynecology, and Reproductive Biology. Jul, 2011  |  Pubmed ID: 21450389

Placental derived vasculogenic/angiogenic substances in maternal blood are dysregulated in pre-eclampsia. We hypothesized that CXCL12, a chemokine with vasculogenic actions, is amongst such molecules.

A Role for Notch Signaling in Trophoblast Endovascular Invasion and in the Pathogenesis of Pre-eclampsia

Development (Cambridge, England). Jul, 2011  |  Pubmed ID: 21693515

Placental trophoblasts (TBs) invade and remodel uterine vessels with an arterial bias. This process, which involves vascular mimicry, re-routes maternal blood to the placenta, but fails in pre-eclampsia. We investigated Notch family members in both contexts, as they play important roles in arterial differentiation/function. Immunoanalyses of tissue sections showed step-wise modulation of Notch receptors/ligands during human TB invasion. Inhibition of Notch signaling reduced invasion of cultured human TBs and expression of the arterial marker EFNB2. In mouse placentas, Notch activity was highest in endovascular TBs. Conditional deletion of Notch2, the only receptor upregulated during mouse TB invasion, reduced arterial invasion, the size of maternal blood canals by 30-40% and placental perfusion by 23%. By E11.5, there was litter-wide lethality in proportion to the number of mutant offspring. In pre-eclampsia, expression of the Notch ligand JAG1 was absent in perivascular and endovascular TBs. We conclude that Notch signaling is crucial for TB vascular invasion.

The Impact of Preeclampsia on Gene Expression at the Maternal-Fetal Interface

Pregnancy Hypertension. Jan, 2011  |  Pubmed ID: 21743843

Preeclampsia (PE) impacts 8 million mother-infant pairs worldwide each year. This human pregnancy-specific disease characterized by hypertension and proteinuria accounts for significant maternal and neonatal morbidity and mortality. The current theory of the pathogenesis of PE as reviewed by Drs. Christopher Redman and Ian Sargent is thought to occur as a 2-stage process with poor placentation in the first half of pregnancy resulting in the maternal response in the second half of pregnancy. Our studies have focused on understanding the placental contribution to this serious disease by examining the gene expression profile of the deciduas basalis or basal plate, the region of the placenta involved in the "poor placentation". In this review we present summaries of our microarray datasets both of normal placentation and those gene expression changes resulting in the context of PE. Additionally, we have taken this opportunity to combine the data sets to provide a more comprehensive view of this region of the placenta. As defects in the basal plate are, in part, at the root of the disease process, we believe that understanding the pathobiology that occurs in this region will increase our ability to alter the development and/or course of PE.

Establishment of Human Trophoblast Progenitor Cell Lines from the Chorion

Stem Cells (Dayton, Ohio). Sep, 2011  |  Pubmed ID: 21755573

Placental trophoblasts are key determinants of in utero development. Mouse trophoblast (TB) stem cells, which were first derived over a decade ago, are a powerful cell culture model for studying their self-renewal or differentiation. Our attempts to isolate an equivalent population from the trophectoderm of human blastocysts generated colonies that quickly differentiated in vitro. This finding suggested that the human placenta has another progenitor niche. Here, we show that the chorion is one such site. Initially, we immunolocalized pluripotency factors and TB fate determinants in the early gestation placenta, amnion, and chorion. Immunoreactive cells were numerous in the chorion. We isolated these cells and plated them in medium containing fibroblast growth factor which is required for human embryonic stem cell self-renewal, and an inhibitor of activin/nodal signaling. Colonies of polarized cells with a limited lifespan emerged. Trypsin dissociation yielded continuously self-replicating monolayers. Colonies and monolayers formed the two major human TB lineages-multinucleate syncytiotrophoblasts and invasive cytotrophoblasts (CTBs). Transcriptional profiling experiments revealed the factors associated with the self-renewal or differentiation of human chorionic TB progenitor cells (TBPCs). They included imprinted genes, NR2F1/2, HMGA2, and adhesion molecules that were required for TBPC differentiation. Together, the results of these experiments suggested that the chorion is one source of epithelial CTB progenitors. These findings explain why CTBs of fully formed chorionic villi have a modest mitotic index and identify the chorionic mesoderm as a niche for TBPCs that support placental growth.

Automated Iterative MS/MS Acquisition: a Tool for Improving Efficiency of Protein Identification Using a LC-MALDI MS Workflow

Analytical Chemistry. Aug, 2011  |  Pubmed ID: 21761829

We have developed an information-dependent, iterative MS/MS acquisition (IMMA) tool for improving MS/MS efficiency, increasing proteome coverage, and shortening analysis time for high-throughput proteomics applications based on the LC-MALDI MS/MS platform. The underlying principle of IMMA is to limit MS/MS analyses to a subset of molecular ions that are likely to identify a maximum number of proteins. IMMA reduces redundancy of MS/MS analyses by excluding from the precursor ion peak lists proteotypic peptides derived from the already identified proteins and uses a retention time prediction algorithm to limit the degree of false exclusions. It also increases the utilization rate of MS/MS spectra by removing "low value" unidentifiable targets like nonpeptides and peptides carrying large loads of modifications, which are flagged by their "nonpeptide" excess-to-nominal mass ratios. For some samples, IMMA increases the number of identified proteins by ∼20-40% when compared to the data dependent methods. IMMA terminates an MS/MS run at the operator-defined point when "costs" (e.g., time of analysis) start to overrun "benefits" (e.g., number of identified proteins), without prior knowledge of sample contents and complexity. To facilitate analysis of closely related samples, IMMA's inclusion list functionality is currently under development.

Comparative Transcriptome Analysis of Human Trophectoderm and Embryonic Stem Cell-derived Trophoblasts Reveal Key Participants in Early Implantation

Biology of Reproduction. Jan, 2012  |  Pubmed ID: 21865555

The implantation process begins with attachment of the trophectoderm (TE) of the blastocyst to the maternal endometrial epithelium. Herein we have investigated the transcriptome of mural TE cells from 13 human blastocysts and compared these with those of human embryonic stem cell (hESC)-derived-TE (hESC(troph)). The transcriptomes of hESC(troph) at Days 8, 10, and 12 had the greatest consistency with TE. Among genes coding for secreted proteins of the TE of human blastocysts and of hESC(troph) are several molecules known to be involved in the implantation process, as well as novel ones, such as CXCL12, HBEGF, inhibin A, DKK3, WNT5A, and follistatin. The similarities between the two lineages underscore some of the known mechanisms and offer discovery of new mechanisms and players in the process of the very early stages of human implantation. We propose that the hESC(troph) is a viable functional model of human trophoblasts to study trophoblast-endometrial interactions. Furthermore, the data derived herein offer the promise of novel diagnostics and therapeutics aimed at practical challenges in human infertility and pregnancy disorders associated with abnormal embryonic implantation.

Breaking the Bottleneck in the Protein Biomarker Pipeline

Clinical Chemistry. Feb, 2012  |  Pubmed ID: 22140214

A Lectin Chromatography/mass Spectrometry Discovery Workflow Identifies Putative Biomarkers of Aggressive Breast Cancers

Journal of Proteome Research. Feb, 2012  |  Pubmed ID: 22309216

We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n = 5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ~90% of which were components of the cell surface or extracellular matrix. We observed 1011 unique glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNAseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were upregulated in triple negative vs. luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.