Two-way communication between endometrial stromal cells and monocytes
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE14808 | GEO | 2009/11/16
SECONDARY ACCESSION(S): PRJNA112041
REPOSITORIES: GEO
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Project description:Endometriosis is an inflammatory disease and bone marrow-derived cells are abundant in endometriotic lesions and in the peritoneal fluid of women with the disease. This study tested the hypothesis that reciprocal communication occurs between macrophages and cultured human endometrial stromal cells and that this communication contributes to the pathology of endometriosis. Changes in gene expression elicited by exposure to factors secreted by the opposing cell type were measured by DNA microarray to test this hypothesis. 716 named genes were differentially expressed in cultured endometrial stromal cells in response to factors secreted by macrophages. Genes that were up-regulated included IL8/CXCL8, MMP3, phospholamban, CYR61/CCN1, CTGF/CCN2, tenascin C, and NNMT, whereas integrin alpha 6 was down-regulated. In contrast, 15 named genes were differentially expressed in macrophages in response to factors secreted by cultured endometrial stromal cells. The data document reciprocal communication between macrophages and endometrial stromal cells and suggest that interaction with macrophages stimulates the expression of genes in endometrial stromal cells that contribute to migration, adhesion, invasion, neovascularization and mitosis of endometrial cells that may support the establishment of endometriosis. Experiment Design: Goal of the experiment: To examine reciprocal communication between a telomerase-immortalized human endometrial stromal cell line (THESC, ATCC #CRL-4003) and U-937 macrophages (ATCC #CRL-1593.2 treated with phorbol ester to stimulate differentiation), and also to assess the response of THESC cells to estrogen plus progesterone. Keywords: human, cultured endometrial stromal cell line, macrophage Experimental factors: hormone treatment and treatment with macrophage-conditioned medium of endometrial stromal cells, treatment of macrophages with endometrial stromal cell conditioned medium Experimental design: T HESC cells were grown in phenol red-free DMEM/F12 medium supplemented with 1% ITS+, 10% charcoal-dextran treated fetal bovine serum albumin, 1% pen/strep, and 500 ng/ml puromycin. THESC cells were treated for 8 days with vehicle (0.1% ethanol) or estradiol (10-8 M) plus progesterone (10-7 M medroxyprogesterone acetate). On day 6, serum was removed from the culture medium. On day 7, half of the cells were treated with U-937 macrophage-conditioned medium for 24 hours. The cells were harvested and RNA extracted on day 8. The experiment was repeated with three different passages of cells (passages 9, 11, and 13) to yield an experimental n of 3. The four groups of cells were vehicle-treated control, estradiol + progesterone, control + macrophage-conditioned medium, and estradiol + progesterone + macrophage-conditioned medium. The monocytic cell line, U-937, was maintained in suspension in RPMI-1640 with 10% fetal bovine serum and 1% pen/strep. The U-937 cells were treated with 32 nM phorbol myristate acetate (PMA) for 48 h to stimulate differentiation into macrophages. The U-937 macrophages were treated with THESC conditioned medium or with non-conditioned control medium. Total RNA was collected after 24 hours. The experiment was repeated with three different passages of cells (passages 7, 9, and 12) to yield an experimental n of 3. The two experimental groups were macrophages treated with non-conditioned control medium and macrophages treated with THESC-conditioned medium. Quality control steps: The cRNA that was synthesized from each cultured cell passage and treatment was used for hybridization to a single CodeLink (Applied Microarrays) whole human genome microarray. Only one sample was hybridized with each slide and only one dye (streptavidin-Alexa 647) was used so no dye swaps were necessary. Bacterial control spikes were used per manufacturer's instructions. Samples used, extract preparation and labelling: The origin of each biological sample: The samples were telomerase-immortalized human endometrial stromal cells and human U-937 monocytic cell line differentiation into macrophages by treatment with phorbol ester. The cell lines were obtained from American Type Culture Collection (#CRL-4003 and #CRL-1593.2). Manipulations of biological samples and protocols used: T HESC cells were grown in culture in phenol red-free DMEM/F12 medium supplemented with 1% ITS+, 2% charcoal/dextran treated fetal bovine serum, 1% penicillin/streptomycin, and 500 ng/ml puromycin. The cells were treated for 8 days with vehicle (0.1% ethanol), estradiol (10-8 M) plus progesterone (10-7 M medroxyprogesterone acetate), vehicle + macrophage-conditioned medium, or estradiol (10-8 M) + progesterone (medroxyprogesterone acetate 10-7 M) + macrophage-conditioned medium. On day 6, serum was removed from the culture medium of all cells. On day 7, half of the cells were treated with macrophage-conditioned medium for 24 hours. The cells were harvested and RNA extracted on day 8. The experiment was repeated with three different passages of cells (passages 9, 11, and 13) to yield an experimental n of 3. The four groups of cells were vehicle-treated control, estradiol + progesterone, control + macrophage-conditioned medium, and estradiol + progesterone + macrophage-conditioned medium. To prepare macrophage-conditioned medium, U-937 (ATCC #CRL-1593.2) cells were grown in RPMI-1640 medium with 10% fetal bovine serum until the cells covered 80% of the flask surface. These U-937 cells were treated with 32 nM PMA for 48 h to stimulate differentiation into macrophages. Differentiated macrophages were maintained in phenol red-free, fetal bovine serum-free DMEM/F12 medium supplemented with 1% penicillin/streptomycin for 48 hours. The cell suspension was centrifuged and the conditioned medium was collected, filtered, and stored at -80C. Before use, macrophage-conditioned medium was supplemented with 1% ITS+ and puromycin. The monocytic cell line, U-937, was maintained in suspension in RPMI-1640 with 10% fetal bovine serum and 1% pen/strep. The U-937 cells were treated with 32 nM PMA for 48 h to stimulate differentiation into macrophages. The differentiated macrophages were treated with THESC conditioned medium or with non-conditioned control medium. Total RNA was collected after 24 hours. Experimental factor: hormone treatment, treatment with macrophage-conditioned medium, and treatment with T-HESC cell conditioned medium Technical protocols: The cells were rinsed and TRI reagent (MRC) was added to the culture flasks. The cells were scraped into centrifuge tubes, bromochloropropane and sodium acetate were added, and the samples were centrifuged to separate the phases. The RNA-containing layer was removed and the RNA purified on an RNeasy extraction column (Qiagen). The sample was treated with an on-column DNase treatment (RNase-free DNase, Qiagen). The purity and quantity were evaluated by an Agilent Bioanalyzer using the RNA 6000 Nanoassay LabChip. Labelled cRNA was prepared using the Message Amp II-Biotin Enhanced Kit from Ambion, following the manufacturer's Instruction Protocol. 1.0 microgram of T-HESC cell total RNA or 2 micrograms of macrophage total RNA was mixed with bacterial control RNA spikes and primed with T7 oligo(dT) primer for 10 min at 70C. (The bacterial control spikes included araB, entF, fixB, gnd, hisB, and leuB.) The first strand of cDNA was synthesized with first strand buffer, dNTP mix, RNase inhibitor, and reverse transcriptase for 2 h at 42C. The second strand cDNA synthesis reaction was prepared using second strand buffer, dNTP mix, DNA polymerase mix, and RNase H; the reaction was carried out for 2h at 16C. The double-stranded cDNA was purified on spin columns included in the Ambion kit. The double-stranded cDNA was mixed with T7 reaction buffer, T7 ATP, T7 GTP, T7 UTP, T7 CTP, biotin-11-UTP, and T7 enzyme mix for the synthesis of cRNA. The cRNA synthesis reaction was terminated after 14h at 37C by purifying the cRNA on spin columns from the Ambion kit. The concentration of cRNA was determined by spectrophotometry. Hybridization procedures and parameters: 10 micrograms of biotinylated cRNA was mixed with fragmentation buffer and heated to 94C for 20 min. The fragmented cRNA was mixed with CodeLink hybridization buffer, loaded on the microarray slides, and hybridized for 18 hours at 37C. The slides were washed in 0.75x TNT (Tris-HCl, NaCl, Tween-20) at 46C for 1h then incubated with streptavidin-Alexa 647 fluorescent dye for 30 min at room temperature. The streptavidin-Alexa 647 fluor was prepared in TNB blocking buffer (0.1M Tris-HCl, 0.15M NaCL, 0.5% NEN Blocking Reagent-PerkinElmer) The slides were then washed 4 times for 5 min each in 1x TNT and twice in 0.05% Tween 20 for 5 sec each. The slides were dried by centrifugation and scanned in an Axon GenePix 4000B scanner.
2010-07-21 | E-GEOD-19834 | biostudies-arrayexpress
Project description:The endometrium contains a distinct population of immune cells consisting of 70% natural killer (NK) cells that undergo cyclic changes during the menstrual cycle. However, how these uterine NK (uNK) cells interact with uterine stromal cells (SC) remains unclear. We therefore investigated the paracrine effect of medium conditioned by uNK cells on the gene expression profile of endometrial SC in-vitro using a cDNA Microarray. Our results, verified by real-time PCR and ELISA, reveal that soluble factors from uNK cells substantially alter endometrial SC gene expression. The largest group of up-regulated genes found were chemokines and cytokines, including IL-15 and IL-15Rα. The latter could produce a niche for uNK cells allowing proliferation within and recruitment into the uterus, as seen in bone marrow. In addition, the most abundantly up-regulated genes, including IL-8, CCL8 and CXCL1 have also been shown to be stimulated by contact of SC with trophoblast, suggesting that uNK cells work synergistically to support the initial trophoblast migration during implantation. Overall this study demonstrates for the first time the paracrine communication between uNK cells and uterine SC, and adds to the understanding of how the uterine immune system contributes to the changes seen within the cycling endometrium. Keywords: Response of endometrial stromal cells to uNK conditioned medium This study was designed to identify the response of non-decidualised stromal cells from the endometrium, to soluble factors secreted by uterine NK cells. Endometrial stromal cells were isolated from 7 patients and treated with control medium or medium conditioned by uterine Nk cells. THE 'REF' COLUMN ON EACH ARRAY IS THE SIGNAL PRODUCED BY A COMMON REFERNCE RNA SAMPLE THAT WAS LABELLED AS A SINGLE BATCH SAMPLE AND HYBRIDISED TO ALL THE ARRAYS- IT IS A 'COMMON REFERENCE'. THE 'TEST' SAMPLE COMPRISES EACH INDIVIDUAL SAMPLE OF CELLS TREATED AS DESCRIBED IN THE SERIES SUBMISSION. FOR EXAMPLE; SAMPLE Y1 (gsm2435820) IS RNA FROM PATIENT 1 TREATED WITH CONTROL MEDIUM, SAMPLE G1 (GSM245371) IS RNA FROM PATIENT 1 TREATED WITH NK CONDITIONED MEDIUM THESE TWO SAMPLES THEREFORE FORM A PAIR- IE CELLS FROM THE SAME PATIENT TREATED WITH CONTROL OR NK-CONDITIONED MIDIUM. Y2,G2 ARE FROM PATIENT 2, ETC
2010-06-24 | E-GEOD-9718 | biostudies-arrayexpress
Project description:Endometriosis is characterized by progesterone resistance and is associated with infertility. KrM-CM-<ppel-like Factor 9 (KLF9) is a progesterone receptor (PGR)-interacting protein, and mice null for Klf9 are subfertile. Whether loss of KLF9 contributes to progesterone resistance of eutopic endometrium of women with endometriosis is unclear. The aim of this study was to investigate KLF9 and PGR co-regulation of human endometrial stromal cell (HESC) transcriptome network. Microarray gene expression analysis was conducted in decidualizing HESC by silencing the expression of KLF9 and PGR, alone or in combination by a siRNA approach, to identify additional KLF9 and PGR co-regulated genes and signaling networks/pathways. HESC also treated with 8-bromo-cAMP, 17M-CM-^_-estradiol, and medroxyprogesterone acetate (cAME) to mimic stromal progression from a proliferative to a differentiated state.
2012-04-03 | E-GEOD-31683 | biostudies-arrayexpress
Project description:The bone marrow microenvironment is a complex mixture of cells that function in concert to regulate hematopoiesis. Cellular components include fixed nonhematopoietic stromal elements as well as monocytes and resident macrophages, which are derived from the hematopoietic stem cells. Although these monocyte-lineage cells are reported to modify stromal cell function, the reverse also occurs. Given the secretory capability of the monocyte/macrophage and their various potential functions, it is not surprising that stromal cells contained within a particular niche can modify monocyte gene expression and functional maturation. Experiment Overall Design: Monocytes were isolated from peripheral blood mononuclear cells from 2 different normal donors and cultured for 48h in conditioned medium (CM). The CM was collected from each of two functionally distinct human bone marrow stromal cell lines (HS5 and HS27a) representing different compartments of the bone marrow microenvironment (Roecklein BA, Torok-Storb B. Blood. 1995;85:997-1005). Four samples were analyzed, with two biological replicates for each CM.
2008-04-06 | E-GEOD-9390 | biostudies-arrayexpress
Project description:Two-way communication between endometrial stromal cells and monocytes
| PRJNA112041 | ENA
Project description:The endometrium contains a distinct population of immune cells consisting of 70% natural killer (NK) cells that undergo cyclic changes during the menstrual cycle. However, how these uterine NK (uNK) cells interact with uterine stromal cells (SC) remains unclear. We therefore investigated the paracrine effect of medium conditioned by uNK cells on the gene expression profile of endometrial SC in-vitro using a cDNA Microarray. Our results, verified by real-time PCR and ELISA, reveal that soluble factors from uNK cells substantially alter endometrial SC gene expression. The largest group of up-regulated genes found were chemokines and cytokines, including IL-15 and IL-15Rα. The latter could produce a niche for uNK cells allowing proliferation within and recruitment into the uterus, as seen in bone marrow. In addition, the most abundantly up-regulated genes, including IL-8, CCL8 and CXCL1 have also been shown to be stimulated by contact of SC with trophoblast, suggesting that uNK cells work synergistically to support the initial trophoblast migration during implantation. Overall this study demonstrates for the first time the paracrine communication between uNK cells and uterine SC, and adds to the understanding of how the uterine immune system contributes to the changes seen within the cycling endometrium. Keywords: Response of endometrial stromal cells to uNK conditioned medium
2009-01-31 | GSE9718 | GEO
Project description:ChickenM-BM- ovalbumin upstream promoter-transcription factor II (COUP-TFII; NR2F2) is an orphan nuclear receptor involved in cell-fate specification, organogenesis, angiogenesis and metabolism. Ablation of COUP-TFII in the mouse uterus causes infertility due to defects in embryo attachment and impaired uterine stromal cell decidualization. Although the function of COUP-TFII in uterine decidualization has been described in mice, its role in the human uterus remains unknown. To better elucidate the mechanisms with which COUP-TFII regulates target gene transcription, genome-wide COUP-TFII binding sites in human endometrial stromal cells (HESC) treated with deciduogenic hormones were identified using ChIP-seq. A total of 16,298 intervals (binding regions) for COUP-TFII were identified compared with the input in HESC chromatin with a very low false discovery rate (0.17%) using a stringent cutoff of p =1x10-10. Distribution of intervals showed that more than half (58.6%) of the COUP-TFII binding sites are located within 10 kb of gene boundaries. 7.5% of total intervals reside within the 10 kb promoter region. A total of 6,077 unique genes were identified to have COUP-TFII binding sites within 10 kb of their gene boundaries. Examination of NR2F2 binding in pooled primary human endometrial stromal cells from 6 healthy women upon decidualization with a hormone cocktail of cAMP, E2 and medroxyprogesterone acetate.
2013-11-02 | E-GEOD-52008 | biostudies-arrayexpress
Project description:The identified stromal factors SDF1alpha, sFRP1 and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells. Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons are potentially useful for treating Parkinson’s disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by co-culture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factor(s) produced by stromal cells that constitute SDIA is unknown. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons in the human embryonal carcinoma stem cell line, NTera2. Here we further examined the effects of PA6-conditioned medium and found that it can induce DA neuronal differentiation in both the NTera2 cell line and the hESC line, I6. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with proteomic analysis of PA6-conditioned medium. Four candidate factors (hepatocyte growth factor (HGF), stromal cell-derived factor-1 alpha (SDF1alpha), secreted frizzled-related protein 1 (sFRP1) and vascular endothelial growth factor D (VEGFD)) were identified and immunoaffinity capillary electrophoresis (ICE) was used to establish the protein concentration of these factors in conditioned medium. Upon addition of SDF1alpha, sFRP1, and VEGFD, we observed an increase in the number of tyrosine hydroxylase- and TuJ1- positive cells in both the NTera2 and I6 cell lines. These results indicate that SDF1alpha, sFRP1 and VEGF-D are major components of SDIA, and suggest the potential use of these defined factors to elicit dopaminergic differentiation of pluripotent stem cells as a therapeutic intervention in PD. Mouse embryonic fibroblasts were grown in DMEM medium supplemented with 10% FBS; this conditioned media was used as the control. PA6, mouse stromal cells, were grown in MEM-alpha supplemented with 10% FBS; this conditioned media is known to induce differentiation in hES cells.
2012-06-24 | E-GEOD-20503 | biostudies-arrayexpress
Project description:All living cells rely on the communication with other cells to ensure their function and survival. Molecular signals are sent among cells of the same cell type and from cells of one cell type to another. In cancer, not only the cancer cells themselves are responsible for the malignancy, but also stromal (non-cancerous) cells and the molecular signals they send to cancer cells are important factors that determine the severity and outcome of the disease. Therefore, the identification of stromal signals and their influence on cancer cells is important when looking for novel treatment strategies. We have generated gene expression profiles of both stromal (HSC), and liver cancer (HCC) cells treated with HSC conditioned medium to elucidate cell communication from HSC to HCC cells. 15 HSC samples, 15 HCC samples treated with conditioned medium of the HSC samples, 4 control hep samples without treatment
2015-04-24 | E-GEOD-62455 | biostudies-arrayexpress
Project description:The identified stromal factors SDF1alpha, sFRP1 and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells. Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons are potentially useful for treating Parkinson’s disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by co-culture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factor(s) produced by stromal cells that constitute SDIA is unknown. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons in the human embryonal carcinoma stem cell line, NTera2. Here we further examined the effects of PA6-conditioned medium and found that it can induce DA neuronal differentiation in both the NTera2 cell line and the hESC line, I6. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with proteomic analysis of PA6-conditioned medium. Four candidate factors (hepatocyte growth factor (HGF), stromal cell-derived factor-1 alpha (SDF1alpha), secreted frizzled-related protein 1 (sFRP1) and vascular endothelial growth factor D (VEGFD)) were identified and immunoaffinity capillary electrophoresis (ICE) was used to establish the protein concentration of these factors in conditioned medium. Upon addition of SDF1alpha, sFRP1, and VEGFD, we observed an increase in the number of tyrosine hydroxylase- and TuJ1- positive cells in both the NTera2 and I6 cell lines. These results indicate that SDF1alpha, sFRP1 and VEGF-D are major components of SDIA, and suggest the potential use of these defined factors to elicit dopaminergic differentiation of pluripotent stem cells as a therapeutic intervention in PD.
2012-06-25 | GSE20503 | GEO