An improved method for the isolation of endometrial epithelial and stromal cells from the total hysterectomy specimen
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE73090 | GEO | 2016/06/14
SECONDARY ACCESSION(S): PRJNA296044
REPOSITORIES: GEO
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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:The prostate stroma is a key mediator of epithelial differentiation and development, and potentially plays a role in the initiation and progression of prostate cancer. Isolation and characterization of viable populations of the constituent cell types of prostate tumors could provide valuable insight into the biology of cancer. The CD90+ stromal fibromuscular cells from tumor specimens were isolated by cell-sorting and analyzed by DNA microarray. Dataset analysis was used to compare gene expression between normal and tumor-associated reactive stromal cells. Reactive stroma is characterized by smooth muscle differentiation, prostate down-regulation of SPOCK3, MSMB, CXCL13, and PAGE4, bladder down-regulation of TRPA1, HSD17B2, IL24, and SALL1, and an up-regulation of CXC-chemokines. This study identified a group of differentially expressed genes in CD90+ reactive stromal cells that are potentially involved in organ development and smooth muscle cell differentiation. Experiment Overall Design: A total of 15 arrays were run for the following sample types obtained from 10 patients: Experiment Overall Design: 2 CD90+ prostate tumor-associated stromal: Experiment Overall Design: Patient 1: CP_Str_08-028_CD90posi Experiment Overall Design: Patient 2: 08-032_CP_strom_CD90posi Experiment Overall Design: 2 CD13+ normal bladder stromal: Experiment Overall Design: Patient 3: 06-125_NB_CD13posi Experiment Overall Design: Patient 4: 06-070_NB_str_CD13posi Experiment Overall Design: 1 CD13+ bladder tumor-associated stromal: Experiment Overall Design: Patient 5: 07-008_CB_str_CD13posi Experiment Overall Design: 5 whole tissue prostate cancer and 5 normal tissue from matched pairs: Experiment Overall Design: Patient 6: 05-206_CaP, 05-206_NP Experiment Overall Design: Patient 7: 05-213_CaP, 05-213_NP Experiment Overall Design: Patient 8: 05-214_CaP, 05-214_NP Experiment Overall Design: Patient 9: 05-218_CaP, 05-218_NP Experiment Overall Design: Patient 10: 05-220_CaP, 05-220_NP Experiment Overall Design: Additionally, 8 arrays were run for the following sample types obtained from 7 patients: Experiment Overall Design: 5 CD49a+ normal prostate stromal (PMID 16638148): Experiment Overall Design: Patient 1: CD49a_01-26-04 Experiment Overall Design: Patient 2: CD49a_03-23-04 Experiment Overall Design: Patient 3: CD49a_03-04-04 Experiment Overall Design: Patient 4: CD49a-1_06-02-04 Experiment Overall Design: Patient 5: CD49a-4_06-02-04 Experiment Overall Design: 3 CD26+ prostate cancer (2 biological replicates, 1 sample run twice): Experiment Overall Design: Patient 6: 05-179_CD26t Experiment Overall Design: Patient 6: 05-179_CD26t_2 Experiment Overall Design: Patient 7: 08-032_CP_epi_CD26posi Experiment Overall Design: The following two prostate cancer samples were also included in the analyses: Experiment Overall Design: CD26+ cancer cell, replicate 1 Experiment Overall Design: CD26+ cancer cell, replicate 2 Experiment Overall Design: The tissue samples consisted of prostate tissue specimens obtained from patients undergoing radical prostatectomy under approval by the University of Washington Institutional Review Board. The same approach was used for both cancer-free and cancer-enriched (where at least 85% of the cells in the corresponding frozen section were of cancer) samples. To obtain bladder stromal cells for analysis, tissue specimens were obtained from cystoprostatectomy surgeries. For cell sorting, the collected specimens were processed within hours. Cell types were sorted using monoclonal antibodies specific for tumor-associated prostate stromal cells (CD90), tumor-associated bladder stromal cells (CD13) and normal bladder stromal cells (CD13) with MACS.
2009-09-18 | E-GEOD-17906 | biostudies-arrayexpress
Project description:To explore the role of small nucleolar RNA (snoRNA) on self-renewal of liver cancer stem cells (CSCs), we isolated liver CSCs (CD133+CD13+) and Non-CSCs (CD133-CD13-) from huamn liver tumor tissues.
2023-06-30 | GSE233521 | GEO
Project description:Primary mesenchymal CD105+CD90+CD13- and CD105+CD90+CD13+ populations from human lung were isolated and characterized using bulk population mRNA sequencing to understand differences between these populations.
2021-12-24 | GSE173738 | GEO
Project description:Two single-cell transcriptional analyses were performed on adult human lung distal and proximal stromal cells, respectively. We isolated lung specimen from both the proximal portion enriched in airways and distal portion enriched in alveoli separately. The lung fragments were then dissociated to single cells and FACSorted for stromal cells based on negative expression of epithelial, hematopoietic, and endothelial markers (EPCAM-/CD45-/CD11b-/CD31-). Library preparation was performed separately for the proximal and distal-derived stromal cells so that each subset has distinctive barcodes to determine anatomical origin, and approximately 10,000 cells were captured from each specimen. The results demonstrate that the proximal and distal human lung stromal subsets demonstrate significant overlap of homologous genes with their murine counterparts generated from Gli2+ murine stroma single-cell transcriptional analysis.
2018-08-08 | GSE108288 | 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:The robust and consistent expression of the CD13 cell surface marker on very early as well as differentiated myeloid hematopoietic cells has prompted numerous investigations seeking to define roles for CD13 in myeloid cells. To directly address the function of myeloid CD13 we created a CD13 null mouse and assessed the responses of purified primary macrophages or dendritic cells from wild type and CD13 null animals in cell assays and inflammatory disease models where CD13 has been previously implicated. We find that mice lacking CD13 develop normally with normal hematopoietic profiles. Moreover, in in vitro assays, CD13 appears to be largely dispensable for the aspects of phagocytosis, proliferation and antigen presentation that we tested, but may contribute to adhesion to endothelial cells. In vivo assessment of four inflammatory disease models showed that lack of CD13 has little effect on disease onset or progression. Nominal alterations in gene expression levels between CD13 wild type and null macrophages argue against compensatory mechanisms. Analysis of the dataset with Ingenuity Pathway Analysis software did not suggest that loss of CD13 resulted in a purturbation of any specific biological pathways, processes or networks. Therefore, while CD13 is highly expressed on myeloid cells and is a reliable marker of the myeloid lineage of both normal and leukemic cells, it is not a critical regulator of hematopoietic development, hemostasis or myeloid cell function. Method: RNA was isolated from resting peritoneal or bone marrow derived macrophages from wild type or CD13 null mice using Trizol according to the manufacturer’s protocol. For quality control, RNA purity and integrity was evaluated by denaturing gel electrophoresis, OD 260/280 ratio and analysis on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Total RNA was amplified and purified using the Ambion Ilumina RNA amplification kit (Ambion, www.ambion.com). Briefly, 300 ng of total RNA was reverse transcribed to cDNA using a T7 oligo(dT) primer. Second-strand cDNA was synthesized, in vitro transcribed and labeled with biotin-16-UTP. The labeled cRNA was analyzed on the Agilent 2100 Bioanalyzer and quantitated by Nanodrop analysis (www.nanodrop.com). The labeled cRNAs were hybridized to the Mouse WholeGenome- 6 BeadChip (Illumina Inc.,) for 16 hours at 58°C, as per manufacturer's instructions. The BeadChips were washed, stained with streptavidin-Cy3 and scanned on the Illumina BeadArray Reader. Analysis: Microarray data was extracted, normalized and analyzed using Illumina GenomeStudio software. Illumina MouseWG-6 v 2.0 Expression BeadChip (Illumina, San Diego, CA) contains 50-mer gene specific oligonucleotide probes corresponding to 45,281 mouse transcript variants. There is on average a 30-fold redundancy for each transcript per array. Intensity data were normalized using the Quantile algorithm in GenomeStudio. Differential expression of each gene, relative to the respective control was evaluated using the Illumina custom error model that calculates a p value as a function of intensity, differential and biological, technical and nonspecific variation. Those samples with p values less than 0.05 were deleted, the relative expression of CD13 null vs. wild type calculated and expressed as fold wild type, and the data ranked from low to high expression relative to wild type values. The dataset was further analyzed using the Ingenuity Pathway Analysis software (Ingenuity Systems, http://www.ingenuity.com).
2010-09-30 | E-GEOD-20985 | biostudies-arrayexpress
Project description:The robust and consistent expression of the CD13 cell surface marker on very early as well as differentiated myeloid hematopoietic cells has prompted numerous investigations seeking to define roles for CD13 in myeloid cells. To directly address the function of myeloid CD13 we created a CD13 null mouse and assessed the responses of purified primary macrophages or dendritic cells from wild type and CD13 null animals in cell assays and inflammatory disease models where CD13 has been previously implicated. We find that mice lacking CD13 develop normally with normal hematopoietic profiles. Moreover, in in vitro assays, CD13 appears to be largely dispensable for the aspects of phagocytosis, proliferation and antigen presentation that we tested, but may contribute to adhesion to endothelial cells. In vivo assessment of four inflammatory disease models showed that lack of CD13 has little effect on disease onset or progression. Nominal alterations in gene expression levels between CD13 wild type and null macrophages argue against compensatory mechanisms. Analysis of the dataset with Ingenuity Pathway Analysis software did not suggest that loss of CD13 resulted in a purturbation of any specific biological pathways, processes or networks. Therefore, while CD13 is highly expressed on myeloid cells and is a reliable marker of the myeloid lineage of both normal and leukemic cells, it is not a critical regulator of hematopoietic development, hemostasis or myeloid cell function.
2010-09-30 | GSE20985 | GEO
Project description:Malignant gliomas are characterized by marked neovascularization and increased tumor cell proliferation. Recently, membrane alanyl-aminopeptidase (CD13/APN) has been identified to play a crucial role in neoangiogenesis. In this study, we show that among various central nervous system tumors, malignant astrocytomas are unique in their high expression levels of functionally active CD13/APN. CD13/APN was found in both tumor cells and tumor vessels of malignant astrocytomas, while in low-grade astrocytomas only endothelial cells of tumor vessels expressed CD13/APN. Inhibitors of the enzymatic activity of CD13/APN significantly reduced the proliferation of U87MG and U138MG malignant glioma cells. Inhibition of CD13/APN mRNA expression by siRNA in glioma cells co-cultured with human umbilical vein endothelial cells (HUVEC) effectively decreased blood vessel formation. Pro-angiogenic factors like bFGF and VEGF induced CD13/APN expression in glioma cells. Treatment of U87MG and U138MG cells with CD13/APN inhibitors resulted in an increased mRNA expression of VEGF and VEGF receptor 2 (VEGF-R2) in these cells. Taken together, these findings provide evidence that CD13/APN promotes tumor cell proliferation and blood vessel formation in malignant astrocytomas. Remarkably, inhibition of CD13/APN induces an angiogenic expression profile via an autocrine feed-back mechanism involving the VEGF/VEGF-R2 system in malignant gliomas.
2012-05-28 | GSE37923 | GEO
Project description:The use of single cell transcriptomics provides previously inaccessible insights into cellular heterogeneity and lineage dynamics of the mammary gland allowing for a better understanding of normal mammary gland function as well as breast cancer initiation and progression. Especially for human mammary gland research, limited tissue accessibility and restriction to ex vivo techniques reinforce the importance of reliable cross-study comparison of single-cell transcriptomic data. However, it is unclear to what extent differences in breast tissue dissociation influence composition and transcriptomic profiles of isolated cells. Here, we used single-cell RNA sequencing to compare human mammary cell populations isolated from a single mammoplasty patient by varying enzymatic dissociation protocols differing in duration (3 or 16 hours) and agitation speed (10 rpm or 100 rpm). Protocol A (3 hours, 100 rpm), protocol B (16 hours, 100 rpm) and protocol C (16 hours, 10 rpm) were used to extract cell fragments from tissue which were either frozen down directly or further dissociated into single cells prior to cryopreservation. Samples were then prepared together for 10x scRNA-sequencing, where the fragments were defrosted and freshly dissociated and were loaded together with the defrosted single cells. From each of the protocols we sequenced similar numbers of cells isolated from fragments (Protocol A: 3,586 cells, Protocol B: 2,809 cells and Protocol C: 4,796 cells), finding an average of 7,427 unique molecular identifiers and 2,445 genes detected per cell. Overall, we detected a greater abundance and heterogeneity of stromal cell types, such as fibroblasts and endothelial cells at a lower agitation speed. Moreover, an extended duration of tissue dissociation governed an overall cellular oxidative stress response together with a downregulation of breast cancer associated genes and a cell-type specific downregulation of lineage markers. Thus, our systematic analysis of dissociation-induced compositional and transcriptional bias in human breast tissue samples yields useful information to avoid misinterpretation of cellular heterogeneity and lineage composition.
2023-06-30 | E-MTAB-9844 | biostudies-arrayexpress