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| Status |
Public on Nov 16, 2009 |
| Title |
Two-way communication between endometrial stromal cells and monocytes |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by array
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| Summary |
Immune system cells and cells of the endometrium have long been proposed to interact in both physiological and pathological processes. The current study was undertaken to examine communication between cultured monocytes and endometrial stromal cells and also to assess responses of endometrial stromal cells to treatment with estradiol (E) in the absence and presence of medroxyprogesterone acetate (P). A telomerase-immortalized human endometrial stromal cell line (T-HESC) and the U-937 monocyte cell line were used. T-HESC were treated with E ± P ± monocyte conditioned medium; U-937 were treated ± T-HESC conditioned medium. Gene expression in response to treatment was examined by DNA microarray. Bi-directional communication, as demonstrated by changes in gene expression, clearly occurred between U-937 monocytes and T-HESC endometrial stromal cells.
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| Overall design |
Experiment Design:
Goal of the experiment: To examine two-way communication between a telomerase-immortalized human endometrial stromal cell line (THESC, ATCC #CRL-4003) and U-937 monocytes (ATCC #CRL-1593.2), and also to assess the response of THESC cells to estrogen in the absence and presence of progesterone.
Brief description of the experiment: Interactions of immune system cells and of the cells of the uterine endometrium have long been proposed to interact in both physiological and pathological processes (i.e., implantation and development of endometriosis). The current study was undertaken to examine communication between cultured monocytes and endometrial stromal cells and also to assess responses of endometrial stromal cells to treatment with estradiol (E) in the absence and presence of medroxyprogesterone acetate (P). A telomerase-immortalized human endometrial stromal cell line (T-HESC) and the U-937 monocyte cell line were used. T-HESC were treated with E ± P ± monocyte conditioned medium; U-937 were treated ± T-HESC conditioned medium. Gene expression in response to treatment was examined by DNA microarray. Bi-directional communication, as demonstrated by changes in gene expression, clearly occurred between U-937 monocytes and T-HESC endometrial stromal cells.
Keywords: human, cultured endometrial stromal cell line, monocyte
Experimental factors: hormone treatment and treatment with monocyte-conditioned medium of endometrial stromal cells, treatment of monocytes 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), estradiol (10-8 M), or with 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 monocyte-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 5, 7, and 9) to yield an experimental n of 3. The six groups of cells were vehicle-treated control, estradiol, estradiol + progesterone, control + monocyte-conditioned medium, estradiol + monocyte-conditioned medium, and estradiol + progesterone + monocyte-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 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). The two experimental groups were non-conditioned control medium and 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 monocytic cells. 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), estradiol (10-8 M) plus progesterone (10-7 M medroxyprogesterone acetate), vehicle + monocyte-conditioned medium, estradiol (10-8 M) + monocyte-conditioned medium, or estradiol (10-8 M) + progesterone (medroxyprogesterone acetate 10-7 M) + monocyte-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 monocyte-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 5, 7, and 9) to yield an experimental n of 3. The six groups of cells were vehicle-treated control, estradiol, estradiol + progesterone, control + monocyte-conditioned medium, estradiol + monocyte-conditioned medium, and estradiol + progesterone + monocyte-conditioned medium.
To prepare monocyte-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. Cells were then 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, monocyte-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 THESC conditioned medium or with non-conditioned control medium. Total RNA was collected after 24 hours.
Experimental factor: hormone treatment, treatment with monocyte-conditioned medium, and treatment with endometrial stromal 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 manufacturer's Instruction Protocol (www1.amershambiosciences.com, CodeLink Gene Expression System: Manual Labelled cRNA Target Preparation). The source of reagents was the CodeLink Expression Assay Reagent Kit, Manual Prep, except where specified otherwise. 1.0 microgram of total endometrial cell RNA or monocytic cell 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 QIAquick columns (Qiagen) and the eluent was dried down in a SpeedVac concentrator. The double-stranded cDNA was resuspended in a mixture containing 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 RNeasy columns (Qiagen). The concentration of cRNA was determined by spectrophotometry.
Hybridization procedures and parameters: 10 micrograms of 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.
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| Contributor(s) |
Eyster KM, Hansen KA, Klinkova O, Mark CJ, Winterton E |
| Citation(s) |
19843877 |
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| Submission date |
Feb 12, 2009 |
| Last update date |
Oct 28, 2014 |
| Contact name |
Kathleen M Eyster |
| E-mail(s) |
Kathleen.Eyster@usd.edu
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| Organization name |
University of South Dakota
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| Department |
Basic Biomedical Sciences
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| Street address |
414 E. Clark St.
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| City |
Vermillion |
| State/province |
SD |
| ZIP/Postal code |
57069 |
| Country |
USA |
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| Platforms (1) |
| GPL2895 |
GE Healthcare/Amersham Biosciences CodeLink Human Whole Genome Bioarray |
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| Samples (24)
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| GSM360054 |
ENDOMETRIAL CELLS ESTRADIOL 2 |
| GSM360055 |
ENDOMETRIAL CELLS ESTRADIOL 3 |
| GSM360056 |
ENDOMETRIAL CELLS ESTRADIOL+PROGESTERONE 1 |
| GSM360057 |
ENDOMETRIAL CELLS ESTRADIOL+PROGESTERONE 2 |
| GSM360058 |
ENDOMETRIAL CELLS ESTRADIOL+PROGESTERONE 3 |
| GSM360059 |
ENDOMETRIAL CELLS ESTRADIOL+PROGESTERONE+MONOCYTE-CONDITIONED MEDIUM 1 |
| GSM360060 |
ENDOMETRIAL CELLS ESTRADIOL+PROGESTERONE+MONOCYTE-CONDITIONED MEDIUM 2 |
| GSM360061 |
ENDOMETRIAL CELLS ESTRADIOL+PROGESTERONE+MONOCYTE-CONDITIONED MEDIUM 3 |
| GSM360062 |
ENDOMETRIAL CELLS ESTRADIOL+MONOCYTE-CONDITIONED MEDIUM 1 |
| GSM360063 |
ENDOMETRIAL CELLS ESTRADIOL+MONOCYTE-CONDITIONED MEDIUM 2 |
| GSM360064 |
ENDOMETRIAL CELLS ESTRADIOL+MONOCYTE-CONDITIONED MEDIUM 3 |
| GSM360065 |
ENDOMETRIAL CELLS VEHICLE+MONOCYTE-CONDITIONED MEDIUM 1 |
| GSM360066 |
ENDOMETRIAL CELLS VEHICLE+MONOCYTE-CONDITIONED MEDIUM 2 |
| GSM360067 |
ENDOMETRIAL CELLS VEHICLE+MONOCYTE-CONDITIONED MEDIUM 3 |
| GSM360068 |
MONOCYTES CONTROL 1 |
| GSM360069 |
MONOCYTES CONTROL 2 |
| GSM360070 |
MONOCYTES CONTROL 3 |
| GSM360071 |
MONOCYTES + THESC CONDITIONED MEDIUM 1 |
| GSM360072 |
MONOCYTES +THESC CONDITIONED MEDIUM 2 |
| GSM360073 |
MONOCYTES + THESC CONDITIONED MEDIUM 3 |
| GSM366623 |
ENDOMETRIAL CELLS CONTROL 2 |
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| Relations |
| BioProject |
PRJNA112041 |
| Supplementary data files not provided |
| Processed data included within Sample table |
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