Tissue-specific Markers Research Articles

Maternal micro-chimeric cells in the multiple sclerosis brain

Maternal microchimeric cells (MMC) pass across the placenta from a mother to her baby during pregnancy. MMC have been identified in healthy adults, but have been reported to be more frequent and at a higher concentration in individuals with autoimmune diseases. MMC in brain tissue from individuals with autoimmune neurological disease has never previously been explored. The present study aims to identify and quantify MMC in adult human brain from control and multiple sclerosis (MS) affected individuals using fluorescent in situ hybridization (FISH) with a probe for the X and Y chromosomes. Post mortem brain tissue from 6 male MS cases and 6 male control cases were examined. Female cells presumed to be MMC were identified in 5/6 MS cases and 6/6 control cases. Cell specific labeling identified female cells of neuronal and immune phenotype in both control and active MS lesion tissue. This study shows that female cells presumed to be MMC are a common phenomenon in adult human brain where they appear to have embedded into brain tissue with the ability to express tissue specific markers.

Baker's yeast (1→3)-β-D-glucan Influences Insulin Sensitivity in Mice with Humanized Obese Diabetic Microbiome in High-Fat Diet-Induced Obesity

AbstractIntroductionβ-glucans are naturally occurring polysaccharides which have isoform specific immunomodulatory and metabolic properties(1). Certain yeast (1→3)-β-D-glucan isoforms improve cholesterol(2), glucose(3) and lipid homeostasis(4). Feeding (1→3)-β-D-glucan alters the microbiome of high-fat diet (HFD) induced obese (DIO)/type 2 diabetic (T2D) mice(5). Here we investigated the potential impact of baker's yeast (1→3)-β-D-glucan in mice humanized with gut microbiomes from either obese healthy versus obese diabetic subjects on immune-metabolism within the context of high-fat feeding.MethodsC57Bl/6J male mice received an antibiotic cocktail of Ampicillin, Metronidazole, Vancomycin, Imipenem and Ciprofloxacin HCl in their drinking water for 6 weeks to diminish the endogenous gut microbiota. Mice were inoculated with microbiota samples obtained from obese healthy (OBH) or diabetic (OBD) humans twice daily for 3 days by oral dosing. Mice were fed a low-fat diet (LFD) (10% kcal) for 4 weeks followed by HFD (45% kcal) with/without baker's yeast (1→3)-β-D-glucan (βG), for 9 weeks. Weight, feed intake, glucose tolerance (1.5g/kg), insulin tolerance (0.5U/kg), hepatic and skeletal lipid levels were examined. Tissue specific molecular markers of metabolism and inflammation, and gut microbiome analysis are being determined to compliment the phenotypic data.ResultsOBH mice were more glucose tolerant and insulin sensitive than OBD mice, despite equal weight gain and adipose tissue mass. Fasting HOMA-IR, attributable to higher insulin concentrations, was higher in OBD compared to OBH mice. βG supplementation reduced HOMA-IR in OBD mice (P < 0.0611). Hepatic triacylglycerol (TAG) and cholesterol levels were also higher in OBD mice, which were prevented by βG supplementation. Hepatic proteomic, caecal microbiomic and metabolomic analysis is on-going in order to ascertain the impact of the OBD versus OBH dysbosis with/without βG supplementation with specific attention on immune-metabolism.

Toxic Effects of Paclobutrazol on Developing Organs at Different Exposure Times in Zebrafish.

To enhance crop productivity and economic profit, farmers often use pesticides that modulate plant growth and prevent disease. However, contamination of ecosystems with agricultural pesticides may impair the health of resident biota. Paclobutrazol (PBZ), an aromatic-containing triazole, is widely applied to many crops in order to promote flowering and fruit setting, while also regulating plant growth and preventing fungus-related diseases. Due to its high mobility, high stability and potential for bioaccumulation, the risks of PBZ to the health of organisms and ecological systems have become a serious concern. In previous studies, we documented the toxicity of PBZ on developing heart, eyes, liver, pancreas and intestine of zebrafish. In this study, we sought to further understand the developmental stage-specific impacts of PBZ on digestive organs and other tissues. Zebrafish were exposed to PBZ beginning at different embryonic stages, and the toxic effects on organs were evaluated at 120 hpf (hours post-fertilization) by in situ hybridization staining with tissue-specific marker genes, such as liver, intestine and pancreas. Unsurprisingly, early-stage embryos exhibited higher sensitivity to PBZ-induced death and developmental hypoplasia of digestive organs. Interestingly, the developing liver and pancreas were more sensitive to PBZ than intestine when embryos were exposed at early stages, but these tissues showed lower sensitivity at later stages. Our delineation of the differential toxic effects of PBZ on developing organs at different exposure timings can serve as a powerful reference for further studies into the mechanisms of PBZ organ toxicity.

Development of Tissue-Specific Age Predictors Using DNA Methylation Data.

DNA methylation patterns have been shown to change throughout the normal aging process. Several studies have found epigenetic aging markers using age predictors, but these studies only focused on blood-specific or tissue-common methylation patterns. Here, we constructed nine tissue-specific age prediction models using methylation array data from normal samples. The constructed models predict the chronological age with good performance (mean absolute error of 5.11 years on average) and show better performance in the independent test than previous multi-tissue age predictors. We also compared tissue-common and tissue-specific aging markers and found that they had different characteristics. Firstly, the tissue-common group tended to contain more positive aging markers with methylation values that increased during the aging process, whereas the tissue-specific group tended to contain more negative aging markers. Secondly, many of the tissue-common markers were located in Cytosine-phosphate-Guanine (CpG) island regions, whereas the tissue-specific markers were located in CpG shore regions. Lastly, the tissue-common CpG markers tended to be located in more evolutionarily conserved regions. In conclusion, our prediction models identified CpG markers that capture both tissue-common and tissue-specific characteristics during the aging process.

A 40-Marker Panel for High Dimensional Characterization of Cancer Immune Microenvironments by Imaging Mass Cytometry.

Multiplex immunophenotyping technologies are indispensable for a deeper understanding of biological systems. Until recently, high-dimensional cellular analyses implied the loss of tissue context as they were mostly performed in single-cell suspensions. The advent of imaging mass cytometry introduced the possibility to simultaneously detect a multitude of cellular markers in tissue sections. This technique can be applied to various tissue sources including snap-frozen and formalin-fixed, paraffin-embedded (FFPE) tissues. However, a number of methodological challenges must be overcome when developing large antibody panels in order to preserve signal intensity and specificity of antigen detection. We report the development of a 40-marker panel for imaging mass cytometry on FFPE tissues with a particular focus on the study of cancer immune microenvironments. It comprises a variety of immune cell markers including lineage and activation markers as well as surrogates of cancer cell states and tissue-specific markers (e.g., stroma, epithelium, vessels) for cellular contextualization within the tissue. Importantly, we developed an optimized workflow for maximum antibody performance by separating antibodies into two distinct incubation steps, at different temperatures and incubation times, shown to significantly improve immunodetection. Furthermore, we provide insight into the antibody validation process and discuss why some antibodies and/or cellular markers are not compatible with the technique. This work is aimed at supporting the implementation of imaging mass cytometry in other laboratories by describing methodological procedures in detail. Furthermore, the panel described here is an excellent immune monitoring tool that can be readily applied in the context of cancer research.

Conserved aging-related signatures of senescence and inflammation in different tissues and species.

Increasing evidence indicates that chronic inflammation and senescence are the cause of many severe age-related diseases, with both biological processes highly upregulated during aging. However, until now, it has remained unknown whether specific inflammation- or senescence-related genes exist that are common between different species or tissues. These potential markers of aging could help to identify possible targets for therapeutic interventions of aging-associated afflictions and might also deepen our understanding of the principal mechanisms of aging. With the objective of identifying such signatures of aging and tissue-specific aging markers, we analyzed a multitude of cross-sectional RNA-Seq data from four evolutionarily distinct species (human, mouse and two fish) and four different tissues (blood, brain, liver and skin). In at least three different species and three different tissues, we identified several genes that displayed similar expression patterns that might serve as potential aging markers. Additionally, we show that genes involved in aging-related processes tend to be tighter controlled in long-lived than in average-lived individuals. These observations hint at a general genetic level that affect an individual’s life span. Altogether, this descriptive study contributes to a better understanding of common aging signatures as well as tissue-specific aging patterns and supplies the basis for further investigative age-related studies.

Diagnostic and prognostic potential of tissue and circulating long non-coding RNAs in colorectal tumors.

Long non-coding RNAs (lncRNAs) are members of the non-protein coding RNA family longer than 200 nucleotides. They participate in the regulation of gene and protein expression influencing apoptosis, cell proliferation and immune responses, thereby playing a critical role in the development and progression of various cancers, including colorectal cancer (CRC). As CRC is one of the most frequently diagnosed malignancies worldwide with high mortality, its screening and early detection are crucial, so the identification of disease-specific biomarkers is necessary. LncRNAs are promising candidates as they are involved in carcinogenesis, and certain lncRNAs (e.g., CCAT1, CRNDE, CRCAL1-4) show altered expression in adenomas, making them potential early diagnostic markers. In addition to being useful as tissue-specific markers, analysis of circulating lncRNAs (e.g., CCAT1, CCAT2, BLACAT1, CRNDE, NEAT1, UCA1) in peripheral blood offers the possibility to establish minimally invasive, liquid biopsy-based diagnostic tests. This review article aims to describe the origin, structure, and functions of lncRNAs and to discuss their contribution to CRC development. Moreover, our purpose is to summarise lncRNAs showing altered expression levels during tumor formation in both colon tissue and plasma/serum samples and to demonstrate their clinical implications as diagnostic or prognostic biomarkers for CRC.

Effects of graphene oxide nanomaterial exposures on the marine bivalve, Crassostrea virginica

Since its discovery in 2004, graphene has been used in a wide variety of fields including biomedicine, electronics, filtration materials, and surface coatings. The rapidly expanding consumer market for graphene family nanomaterials (GFNs), such as graphene oxide (GO), raises concern regarding their environmental toxicity. The aim of this study was to evaluate the effects of GO exposures in a marine filter-feeding bivalve (Crassostrea virginica) using sublethal biomarker approaches that can contribute to the development of an adverse outcome pathway (AOP). A 14-day study was conducted to identify tissue-specific molecular markers of GO toxicity using a static renewal design. Elevated lipid peroxidation and changes in glutathione-s-transferase (GST) activities were observed in gills and digestive gland tissues of the GO-exposed oysters. These cellular changes were noted for 2.5 and 5 mg/L GO exposures in seawater. Based on our results, reactive oxygen species (ROS)-induced oxidative damage is identified as a key event in the proposed AOP. Additionally, detoxification enzymes, such as GST, are thought to be involved in stress signaling leading to adverse effects on cellular health. This study is a part of our two-tier approach towards the identification of short- and long-term effects of GO exposures. This work, together with our previous 72 h exposure, represents the application of biomarker-based investigations in the process of AOP development for graphene family nanomaterials.

Preparation of cfMeDIP-seq libraries for methylome profiling of plasma cell-free DNA

Circulating cell-free DNA (cfDNA) comprises small DNA fragments derived from normal and tumor tissue that are released into the bloodstream. Recently, methylation profiling of cfDNA as a liquid biopsy tool has been gaining prominence due to the presence of tissue-specific markers in cfDNA. We have previously reported cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) as a sensitive, low-input, cost-efficient and bisulfite-free approach to profiling DNA methylomes of plasma cfDNA. cfMeDIP-seq is an extension of a previously published MeDIP-seq protocol and is adapted to allow for methylome profiling of samples with low input (ranging from 1 to 10 ng) of DNA, which is enabled by the addition of 'filler DNA' before immunoprecipitation. This protocol is not limited to plasma cfDNA; it can also be applied to other samples that are naturally sheared and at low availability (e.g., urinary cfDNA and cerebrospinal fluid cfDNA), and is potentially applicable to other applications beyond cancer detection, including prenatal diagnostics, cardiology and monitoring of immune response. The protocol presented here should enable any standard molecular laboratory to generate cfMeDIP-seq libraries from plasma cfDNA in ~3-4 d.

Differential regulation of auxin and cytokinin during the secondary vascular tissue regeneration in Populus trees.

Tissue regeneration upon wounding in plants highlights the developmental plasticity of plants. Previous studies have described the morphological and molecular changes of secondary vascular tissue (SVT) regeneration after large-scale bark girdling in trees. However, how phytohormones regulate SVT regeneration is still unknown. Here, we established a novel in vitro SVT regeneration system in the hybrid aspen (Populus tremula × Populus tremuloides) clone T89 to bypass the limitation of using field-grown trees. The effects of phytohormones on SVT regeneration were investigated by applying exogenous hormones and utilizing various transgenic trees. Vascular tissue-specific markers and hormonal response factors were also examined during SVT regeneration. Using this in vitro regeneration system, we demonstrated that auxin and cytokinin differentially regulate phloem and cambium regeneration. Whereas auxin is sufficient to induce regeneration of phloem prior to continuous cambium restoration, cytokinin only promotes the formation of new phloem, not cambium. The positive role of cytokinin on phloem regeneration was further confirmed in cytokinin overexpression trees. Analysis of a DR5 reporter transgenic line further suggested that cytokinin blocks the re-establishment of auxin gradients, which is required for the cambium formation. Investigation on the auxin and cytokinin signalling genes indicated these two hormones interact to regulate SVT regeneration. Taken together, the in vitro SVT regeneration system allows us to make use of various molecular and genetic tools to investigate SVT regeneration. Our results confirmed that complementary auxin and cytokinin domains are required for phloem and cambium reconstruction.

Preliminary results indicate increased expression of miR-184 in patients with renal carcinoma.

Renal carcinoma is the second most common cancer in the urinary system with an increasing trend. The major treatment for renal carcinoma is surgery, which results in unfavorable prognosis at times. As a tissue-specific marker for tumor, microRNA (miR) exerts its functions via facilitating oncogenic gene expression or suppressing tumor suppressor gene. MiR-184 is known to be abnormally expressed in various tumors. There are few studies about the lack of miR-184 expression in renal carcinoma. Real time-Polymerase Chain Reaction (PCR) was used to measure the expression of miR-184 in 38 renal carcinoma and adjacent tissues. The in vitro cultured renal carcinoma cell line ACHN was transfected with miR-184 mimic or inhibitor. The expression of miR-184 was measured by real time-PCR, and the cell proliferation was measured by MTT assay. The cell colony formation was examined, and the cell invasion potency was assessed by transwell assay. The apoptotic activity was measured by flow cytometry, and the Western blot detected protein expression change of β-catenin/TCF3 pathway. Compared to tumor-adjacent tissues, miR-184 and β-catenin/TCF3 showed an elevated expression in renal carcinoma tissues which were further increased with elevated RC stages (p<0.05). The transfection of miR-184 mimic into ACHN cells increased its expression, enhanced ACHN cell proliferation, colony formation, inhibited apoptosis, promoted tumor cell invasion, and increased the expression of β-catenin and TCF4 proteins (p<0.05 compared to NC control group). MiR-184 is up-regulated in renal carcinoma tissues. The downregulation of miR-184 in renal carcinoma cells could facilitate cell apoptosis and inhibited tumor proliferation or invasion possibly via modulating β-catenin/TCF4 pathway.

Humulus japonicus stimulates thermogenesis and ameliorates oxidative stress in mouse adipocytes.

An aqueous extract of Humulus japonicus (AH) has been documented to ameliorate hypertension and non-alcoholic fatty liver disease (NAFLD). Here, we investigated the effects of an aqueous extract of AH on thermogenesis and palmitate-induced oxidative stress in adipocytes. To verify the effect of AH on browning, we measured the expression levels of specific markers in 3T3-L1 adipocytes using qPCR and Western blotting, respectively. To assess the role of oxidative stress, cells were stained with DCFDA and observed by fluorescence microscopy. AH increased the expression of brown adipose tissue-specific markers. Additionally, it induced fatty acid oxidation and lipolysis and suppressed both lipogenic markers and lipid accumulation. Furthermore, AH ameliorated hydrogen peroxide-induced oxidative stress. Enhanced expression of these markers contributed to fat browning, fatty acid oxidation and lipolysis of 3T3-L1 adipocytes via the AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor delta (PPARδ) signaling pathways. Moreover, AMPK and PPARδ resulting in protective effects of AH against oxidative stress. In sum, AH could promote the browning, lipolysis and thermogenesis in 3T3-L1 adipocytes and would suppress the hydrogen peroxide-induced oxidative stress and lipogenesis during differentiation. We therefore suggest that AH could be used as a potential candidate for treating obesity and related metabolic disorders.

Comprehensive DNA methylation analysis of tissue of origin of plasma cell-free DNA by methylated CpG tandem amplification and sequencing (MCTA-Seq)

BackgroundComprehensive analysis of the tissue of origin of plasma cell-free DNA (cfDNA) remains insufficient. A genome-scale DNA methylation method for this analysis is of both biological and clinical interest.MethodsWe used the methylated CpG tandem amplification and sequencing (MCTA-Seq), which is a genome-scale DNA methylation method, for analyzing cfDNA. We performed MCTA-Seq to pair plasma cfDNA and white blood cell genomic DNA from 14 healthy individuals for comparative analysis, with eight tissues being analyzed for identifying tissue-specific markers. The relative contributions of multiple tissues to cfDNA were calculated for plasma cfDNA obtained from healthy adults (n = 25), cholelithiasis patients (n = 13), liver cirrhosis patients (n = 17), hepatocellular carcinoma patients (n = 30), and acute pancreatitis patients (n = 8).ResultsWe identified a total of 146 tissue-specific hypermethylation markers. Simulation analysis showed that MCTA-Seq can accurately measure DNA fractions contributed by multiple tissues to cfDNA. We demonstrated that the liver is the major non-hematopoietic tissue contributing to plasma cfDNA in healthy adults. The method also detected increases in the liver-derived DNA in the blood from patients with liver diseases, which correlate with an increase in the liver enzyme level. Furthermore, the results indicated that blood cells make a major contribution to the elevation of cfDNA levels in acute pancreatitis, liver cirrhosis, and hepatocellular carcinoma patients. Finally, we characterized a novel set of tissue-specific hypermethylation markers for cfDNA detection, which are located within the intragenic regions of tissue-specific highly expressed genes.ConclusionsWe have used MCTA-Seq for simultaneously measuring cfDNA fractions contributed by multiple tissues. Applying this approach to healthy adults and liver and pancreas disease patients revealed the tissue of origin of cfDNA. The approach and the identified markers should facilitate assessing the cfDNA dynamics in a variety of human diseases.

Trends in Colorectal Cancer Surveillance: Current Strategies and Future Innovations-

This review article strives to reflect on the historic surveillance debate in colorectal cancer, outline current strategies, and guidelines, and discuss new techniques being explored in surveilling colorectal adenocarcinoma. Within the past decade, major governing bodies have proposed surveillance guidelines with the goal of earlier identification of cancer recurrence, thereby possibly reducing the morbidity and mortality of necessary interventions. With the innovation of tissue-specific tumor markers and fluorescence endoscopy, the approach to surveillance may be changing. The use of these new modalities allows clinicians to provide a more risk-adjusted basis for care, which is predicted to equate to higher quality care. The current surveillance guidelines provide an evidence-based framework for physicians and surgeons to follow. However, the influence of these novel surveillance techniques in colorectal cancer is yet to be realized and they ultimately have the potential to revolutionize care.

Adipocyte lineage differentiation potential of MSCs isolated from reaming material.

Mesenchymal stem cells (MSCs) obtained from various sources have been used for different therapeutic applications including tissue regeneration. Reamer/irrigator/aspirator (RIA) has been increasingly used in recent years for the derivation of MSCs. Here in this investigation we have comparatively analyzed MSCs obtained from iliac crest bone marrow (ICBM) and RIA for their morphology, cluster determinant (CD) markers, and adipogenic differentiation capacity. MSCs were isolated, cultured, and purified from both sources and then flow cytometric studies were performed to study their characteristics. The differentiation potential of RIA and ICBM was examined by an Oil Red O staining protocol. Moreover, the tissue-specific markers related to adipogenesis were analyzed by real-time polymerase chain reaction (RT-PCR). The cells were cultured in the relevant induction medium and then adipogenic lineage differentiation was tested and confirmed for all MSC preparations. Additionally, analysis by flow cytometer was indicative of RIA derived MSCs (RIA-MSCs) having a more homogenous population than ICBM derived MSCs. The RIA-MSCs differentiation toward adipogenic lineage was more efficient compared with ICBM-MSCs. Direct comparative analysis of RIA to ICBM-MSCs indicated that the RIA-MSCs had a higher potential toward adipocyte lineage differentiation compared with ICBM-MSCs.

Donor T and NK Cells are Derived from the Donor Lung Parenchyme and Represent Tissue-Resident Memory Cells - An Important Feature for Tolerance Development

Purpose In previous studies, we identified donor lymphocytes in peripheral blood of recipients directly after transplantation and persisting at least three weeks that were characterized by the tissue retention marker CD69. In order to determine their origin, we hypothesized that donor T and NK cells have a peculiar tissue-resident memory phenotype that is shared between cells derived from lung perfusates, trachea parenchyma and lymph nodes. Methods Donor lymphocytes in recipient blood were determined in 27 lung transplant patients at T0, T24, 3 weeks by staining of donor HLA class I molecules in combination with lineage- and tissue-specific markers using flow cytometry. The phenotype of T and NK cells in perfusates (n=30), donor trachea (n=5), and lymph node (n=10), recipient explanted parenchyma (n=15) was compared to circulating cells using the same markers. Results In peripheral blood of all lung transplant recipients, donor derived T and NK cells were detected at T0, T24 and 3 weeks and had higher CD69 expression compared to recipient cells (p=0.001 to 0.03) and were mostly CD25−. This phenotype was similar to T and NK cells in corresponding perfusates, with significantly increased CD69 expression compared to circulating PBMCs (all p Conclusion Our results suggest that donor T and NK cells found in the periphery of lung transplant recipients are derived from lung parenchyma and represent tissue-resident memory cells. This transient chimerism in recipient blood might be clinically relevant for tolerance induction after transplantation due to unique features of TRM T and NK cells.

Compact buds with biphasic differentiation and calcitonin-expressing neuroendocrine cells-previously unrecognized structures of thyroglossal duct unveiled by immunohistochemistry.

Immunophenotype of thyroglossal duct (TGD) cysts, including lining epithelium and thyroid remnants, is scarcely addressed in the literature. There is indirect evidence that C cells may be derived from progenitor cells of the midline thyroid primordium. This is supported by the recent concept of the endodermal origin of lateral thyroid anlagen and several case reports. We aimed to search for neuroendocrine cells in TGD cysts and to characterize immunophenotype of the thyroid follicles and epithelial lining of TGD. Out of 98 TGD cysts, 70% contained both cyst-lining epithelium and thyroid follicles, whereas 30% possessed only cyst-lining epithelium. Specimens eligible for immunohistochemistry (n = 61) were stained for thyroid-specific and neuroendocrine markers. Thyroid remnants were positive for thyroid transcription factor 1 (TTF-1) and other thyroid tissue-specific markers and negative for calcitonin. TGD epithelium showed strong p63 positivity. We found that respiratory epithelium in 9.8% of TGDs contained neuroendocrine cells positive for calcitonin, chromogranin A, and synaptophysin but negative for carcinoembryonic antigen. In 44.2% of the cases, we detected compact buds, microscopic structures appearing as nests of epithelial cells with a biphasic population of basal (p63+) and central (TTF-1+) cells. Thyroid remnants in TGD expressed full spectrum of thyroid-specific markers and contained no C cells. Instead, calcitonin-expressing neuroendocrine cells were found among the respiratory epithelium of TGD. These cells can be a potential source of neuroendocrine tumors mimicking medullary carcinoma in median anlage derivatives. We also discovered precursor compact buds with dual immunophenotype and proposed a concept of their morphogenesis.

Nanoscale flow cytometry to distinguish subpopulations of prostate extracellular vesicles in patient plasma.

To determine if prostate-derived extracellular vesicles (EVs) present in patient plasma samples are of exocytotic origin (exosomes) or released by the cell membrane (microparticles/microvesicles). Both malignant and normal prostate cells release two types of EVs into the circulation, exosomes, and microparticles/microvesicles which differ in size, origin, and mode of release. Determining what proportion of prostate-derived EVs are of exosomal versus microparticle/microvesicle EV subtype is of potential diagnostic significance. Multi-parametric analytical platforms such as nanoscale flow cytometry (nFC) were used to analyze prostate derived extracellular vesicles. Plasmas from prostate cancer (PCa) patient plasmas representing benign prostatic hyperplasia (BPH), low grade prostate cancer (Gleason Score 3 + 3) and high grade prostate cancer (Gleason Score ≥4 + 4) were analyzed for various exosome markers (CD9, CD63, CD81) and a prostate specific tissue marker (prostate specific membrane antigen/PSMA). By using nanoscale flow cytometry, we determine that prostate derived EVs are primarily of cell membrane origin, microparticles/microvesicles, and not all PSMA expressing EVs co-express exosomal markers such as CD9, CD63, and CD81. CD9 was the most abundant exosomal marker on prostate derived EVs (12-19%). There was no trend observed in terms of more PSMA + CD9 or PSMA + CD63 co-expressing EVs versus increasing grade of prostate cancer. The majority of prostate derived EVs present in plasmas are from the cell membrane as evidenced by their size and most importantly, lack of co-expression of exosomal markers such as CD9/CD63/CD81. In fact, CD81 was not present on any prostate derived EVs in patient plasmas whereas CD9 was present on a minority of prostate derived EVs. The addition of an exosomal marker for detection of prostate-derived EVs does not provide greater clarity in distinguishing EVs released by the prostate.

Isolation and characterization of ovine umbilical cord-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are able to self-renew and have multi-lineage differentiation potential. However, studies on ovine umbilical cord-derived MSCs (UC-MSCs) are limited. Our study aimed to isolate and characterize ovine UC-MSCs. We successfully isolated ovine UC-MSCs and defined their surface marker profile using immunofluorescence analysis. Ovine UC-MSCs were found to be positive for cell surface markers CD13, CD29, CD44, CD90, and CD106, and negative for cell surface marker CD45. Assessment of the proliferation potential of ovine UC-MSCs showed that from day 3 of cultivation a plateau phase was reached. And compare to passage 10, 15, 20 cells, passage 5 cells proliferating the fastest. Differentiation of ovine UC-MSCs into adipocytes, osteocytes, and chondrocytes was also demonstrated by staining for tissue-specific markers and using quantitative real-time polymerase chain reaction for specific marker gene expression. This study demonstrates the existence of a MSC population within the ovine umbilical cord, which maintained a normal karyotype up to passage 20.

Integrated Eutopic Endometrium and Non-Depleted Serum Quantitative Proteomic Analysis Identifies Candidate Serological Markers of Endometriosis.

Endometriosis affects about 4% of women in the reproductive age and is associated with subfertility. The aim of the present study is to examine the integrated quantitative proteomic profile of eutopic endometrium and serum from women with endometriosis compared to controls in order to identify candidate disease-specific serological markers. Eutopic endometrium and serum from patients with endometriosis (n = 8 for tissue and n = 4 for serum) are, respectively, compared to endometrium and serum from females without endometriosis (n = 8 for tissue and n = 4 for serum) using a shotgun quantitative proteomics method. All study participants are at the proliferative phase of their menstrual cycle. At the tissue and serum level, 1214 and 404 proteins are differentially expressed (DEPs) in eutopic endometrium and serum, respectively, of women with endometriosis versus controls. Gene ontology analysis shows that terms related to immune response/inflammation, cell adhesion/migration, and blood coagulation are significantly enriched in the DEPs of eutopic endometrium, as well as serum. Twenty-one DEPs have the same trend of differential expression in both matrices and can be further examined as potential disease- and tissue-specific serological markers of endometriosis. The present integrated proteomic profiling of eutopic endometrium and serum from women with endometriosis identify promising serological markers that can be further validated in larger cohorts for the minimally invasive diagnosis of endometriosis.