Homo sapiens
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA296044 | ENA |
REPOSITORIES: ENA
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Project description:We aimed to improve the efficiency of isolating endometrial epithelial and stromal cells (EMECs and EMSCs) from hysterectomy specimens. We revealed by immunohistochemical staining that the large tissue fragments remaining after collagenase treatment, which are usually discarded after the first filtration in the conventional protocol, consisted of glandular epithelial and stromal cells. Therefore, we established protease-treatment and cell suspension conditions to dissociate single cells from the tissue fragments, and isolated epithelial (EPCAM-positive) and stromal (CD13-positive) cells by fluorescence-activated cell sorting. Four independent experiments showed that, on average, 1.2 x 10^6 of EMECs and 2.8 x 10^6 EMSCs were isolated from one hysterectomy specimen. We confirmed that the isolated cells presented transcriptomic features highly similar to those of epithelial and stromal cells obtained by the conventional method. Our improved protocol facilitates future studies to better understand the epigenetic regulation underlying the dynamic changes of the endometrium during the menstrual cycle. Although protocols for the isolation of endometrial stromal and epithelial cells (EMSCs and EMECs) have been well established, the number of EMECs obtainable by the current protocols is relatively small. To improve the efficiency of isolating EMECs as well as EMSCs from endometrial tissues, we established protease-treatment and cell suspension conditions to efficiently dissociate single cells from endometrial tissue fragments, and isolated epithelial (EPCAM-positive) and stromal (CD13-positive) cells by fluorescence-activated cell sorting. By conducting a microarray-based transcriptome analysis, we confirmed that the cells isolated by the modified protocol developed in this study maintain the transcriptomic properties of endometrial epithelial and stromal cells.
2016-06-14 | E-GEOD-73090 | biostudies-arrayexpress
Project description:In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation in Low Grade Endometrial Stromal Sarcoma (LG-ESS) and Ossifying FibroMyxoid Tumors (OFMT). We express the fusion protein and necessary controls in K562 Cells. The fusion protein assembles a mega-complex harboring both NuA4/TIP60 and PRC2 subunits and enzymatic activities and leads to mislocalization of chromatin marks in the genome, linked to aberrant gene expression.
2020-12-03 | GSE162543 | GEO
Project description:In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation in Low Grade Endometrial Stromal Sarcoma (LG-ESS) and Ossifying FibroMyxoid Tumors (OFMT). We express the fusion protein and necessary controls in K562 Cells. The fusion protein assembles a mega-complex harboring both NuA4/TIP60 and PRC2 subunits and enzymatic activities and leads to mislocalization of chromatin marks in the genome, linked to aberrant gene expression.
2020-11-25 | GSE162118 | GEO
Project description:We aimed to improve the efficiency of isolating endometrial epithelial and stromal cells (EMECs and EMSCs) from hysterectomy specimens. We revealed by immunohistochemical staining that the large tissue fragments remaining after collagenase treatment, which are usually discarded after the first filtration in the conventional protocol, consisted of glandular epithelial and stromal cells. Therefore, we established protease-treatment and cell suspension conditions to dissociate single cells from the tissue fragments, and isolated epithelial (EPCAM-positive) and stromal (CD13-positive) cells by fluorescence-activated cell sorting. Four independent experiments showed that, on average, 1.2 x 10^6 of EMECs and 2.8 x 10^6 EMSCs were isolated from one hysterectomy specimen. We confirmed that the isolated cells presented transcriptomic features highly similar to those of epithelial and stromal cells obtained by the conventional method. Our improved protocol facilitates future studies to better understand the epigenetic regulation underlying the dynamic changes of the endometrium during the menstrual cycle.
2016-06-14 | GSE73090 | GEO
Project description:In this study, we characterize the fusion protein produced by the EPC1-PHF1 translocation in Low Grade Endometrial Stromal Sarcoma (LG-ESS) and Ossifying FibroMyxoid Tumors (OFMT). We express the fusion protein and necessary controls in K562 Cells. The fusion protein assembles a mega-complex harboring both NuA4/TIP60 and PRC2 subunits and enzymatic activities and leads to mislocalization of chromatin marks in the genome, linked to aberrant gene expression.
2022-02-18 | GSE196754 | GEO
Project description:Endometrial epithelial cell transcriptome response to co-culture with adipose stromal cells
| PRJNA400484 | ENA
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
| S-EPMC2531291 | biostudies-literature
Project description:RNA extracted at 240min. Samples are rRNA depleted. Expression measurement of Homo sapiens genes.
2019-07-31 | GSE114179 | GEO
Project description:During reproductive life, the human endometrium undergoes around 480 cycles of growth, breakdown and regeneration should pregnancy not be achieved. This outstanding regenerative capacity is the basis for women’s cycling and its dysfunction may be involved in the etiology of pathological disorders. Therefore, the human endometrial tissue must rely on a remarkable endometrial somatic stem cells (SSC) population. Here we explore the hypothesis that human endometrial side population (SP) cells correspond to somatic stem cells. We isolated, identified and characterized the SP corresponding to the stromal and epithelial compartments using endometrial SP genes signature, immunophenotyping and characteristic telomerase pattern. We analyzed the clonogenic activity of SP cells under hypoxic conditions and the differentiation capacity in vitro to adipogenic and osteogenic lineages. Finally, we demonstrated the functional capability of endometrial SP to develop human endometrium after subcutaneous injection in NOD-SCID mice. Briefly, SP cells of human endometrium from epithelial and stromal compartments display genotypic, phenotypic and functional features of SSC. Human tissue collection: Human endometrial biopsies were taken using a Pipelle catheter under sterile conditions (from 18 to 48 years) throughout the menstrual cycle. Epithelial and stromal separation: Epithelial and stromal fractions were isolated using an established protocol with modifications. Briefly, samples were carefully dissected and minced into 1-2mm fragments, and enzymatically digested in DMEM containing 10 mg/ml collagenase type IA. Stromal cells (single cells or small aggregates) and epithelial glands were separated on a size basis using gravity sedimentation and membrane filtration. Cell suspensions were treated with erythrocyte lysis solution and evaluation of cell viability was performed with Propidium Iodide (PI; 5 μg/ml). Hoechst 33342 labeling: Cells isolated from the endometrial epithelial and stromal fractions were resuspended in DMEM prewarmed at 37ºC and supplemented with 2% FBS and 10mM HEPES. The cell suspension was labeled in the same media with 5 μg/ml of Hoechst 33342 dye (Ho-33342), either alone or in combination with 100 μM verapamil (Vp), in a water bath at 37ºC for 90-120 minutes. Then, cells were centrifuged for 6 minutes at 4ºC, and were resuspended in cold HBSS supplemented with 2%FBS and 10mM HEPES. PI permits to exclude dead cells prior to the flow cytometric analysis and sorting. Isolation of human endometrial SP cells: Cells were analyzed and sorted by a MoFlo® jet-in-air high speed sorter. Excitation was performed with a water cooled Enterprise II ion laser which operated at the 351 nm and 488 nm wavelengths, and worked at 30 mW. Hoechst 33342 blue and red fluorescences were detected through 405/30 and 670/20 nm band-pass filters respectively, by measuring the signals on a linear scale. PI fluorescence was detected through a band-pass filter of 613/20 on a logarithmic scale. The gates for cell sorting were defined to collect live cells with a low Hoechst 33342 fluorescence (SP fraction), as well as live cells with a high Hoechst 33342 fluorescence (NSP fraction). Microarrays experiments: Endometrial tissue consisted in a total of 8 endometrial biopsies (epithelial (n=8) and stromal (n=8) endometrial cell suspensions, epithelial SP fraction (n=8) and stromal SP fractions (n=8) separately) which were pooled in pairs at the RNA levels. Four microarrays were analyzed per group.
2010-08-15 | E-GEOD-21633 | biostudies-arrayexpress
Project description:We present a method for identifying and ascribing differentially expressed genes to tumor epithelial and/or stromal cells, by utilizing pathologic information and weighted t-statistics Sets of differentially expressed gene-probes were identified in tumors from patients who developed distant metastasis compared with those who did not, by weighing the contribution from each tumor with the relative content of stromal and tumor epithelial cells in their individual tumor specimen. The analyses were performed under various assumptions of mRNA transcription level from tumor epithelial cells compared with stromal cells. Supplementary files: Human_AB1700_Annotations_09_06.txt: This file includes control probes and obsolete probes not listed in GPL2986. Raw_data.txt: This file includes control probes not listed in GPL2986.
2010-09-13 | E-GEOD-24117 | biostudies-arrayexpress