Human endometrial side population cells exhibit genotypic, phenotypic and functional features of somatic stem cells.
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ABSTRACT:
SUBMITTER: Cervello I
PROVIDER: S-EPMC2891991 | biostudies-literature | 2010 Jun
REPOSITORIES: biostudies-literature
Publications
Human endometrial side population cells exhibit genotypic, phenotypic and functional features of somatic stem cells.
Cervelló Irene I Gil-Sanchis Claudia C Mas Aymara A Delgado-Rosas Francisco F Martínez-Conejero José Antonio JA Galán Amparo A Martínez-Romero Alicia A Martínez Sebastian S Navarro Ismael I Ferro Jaime J Horcajadas José Antonio JA Esteban Francisco José FJ O'Connor José Enrique JE Pellicer Antonio A Simón Carlos C
PloS one 20100624 6
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 corre ...[more]
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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: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.
2010-08-15 | GSE21633 | GEO
Project description:To sum up our results, we managed to isolate a reproducible SP cell fraction of human gastric carcinoma cell lines for the first time. Those cells differed from the rest of the population regarding morphology and biological behaviour. They had the ability to build a population resembling the basic tumor population and therefore performed differenziation and asymmetric cell division. SP cells expressed potential stem cell markers like musashi 1 and Cd133 and also showed a high expression of genes that encode for stem cell properties. The so identified phenotypic and genetic factors showed a variable expression in tumor samples of patient series and might be applicable as prognostic factors in the diagnosis of gastric carcinoma.
2010-12-01 | GSE20058 | GEO
Project description:To sum up our results, we managed to isolate a reproducible SP cell fraction of human gastric carcinoma cell lines for the first time. Those cells differed from the rest of the population regarding morphology and biological behaviour. They had the ability to build a population resembling the basic tumor population and therefore performed differenziation and asymmetric cell division. SP cells expressed potential stem cell markers like musashi 1 and Cd133 and also showed a high expression of genes that encode for stem cell properties. The so identified phenotypic and genetic factors showed a variable expression in tumor samples of patient series and might be applicable as prognostic factors in the diagnosis of gastric carcinoma. Aims: The side population (SP) of tumor cell lines shares characteristics with tumor stem cells. In this study we phenotypically and genotypically characterized the SP of gastric cancer cell lines. Methods: The SP was obtained from MKN45- and AGS-gastric cancer cells using Hoechst 33342 staining and fluorescence-activated cell sorting (FACS). SP cells were subsequently studied morphologically (cytology, immunocytochemistry), on the transcriptional level (gene array) and in cell culture (recultivation assays). Genes differentially expressed in the SP cells were finally searched by immunohistochemistry in neoplastic and non-neoplastic gastric tissue from gastric cancer patients. Results: The SP was reproducibly obtained from gastric cancer cell lines. The SP cells were smaller and rounder then non-SP cells. SP cells self-renewed in re-cultivation experiments and differentiated into SP- and non-SP cells. Re-cultivated SP- and non-SP cells showed distinct phenotypes in culture regarding cell shape and colony-formation. SP cells had increased levels of the stem cell markers CD133 and Musashi 1. Transcriptional analyses demonstrated that SP cells express genes that encode for stem cell properties like FZD7, HEY1, SMO and ADAM17. Finally we detected the transcripts of these genes in tissue samples from patients with gastric cancer. Conclusions: Human gastric cancer cell lines enclose a phenotypically and genotypically distinct cell population with tumor stem cell features. Phenotypical characteristics of this distinct cell population are also present in gastric cancer tissue.
2010-12-01 | E-GEOD-20058 | biostudies-arrayexpress
Project description:The side population (SP) of tumor cell lines shares characteristics with tumor stem cells. The objective of this study was to phenotypically and genotypically characterize the SP of gastric cancer cell lines. SP cells were obtained from AGS and MKN45 gastric cancer cells using Hoechst 33342 staining and fluorescence-activated cell sorting. The cells were subsequently studied morphologically at cytology and immunocytochemistry, on the transcriptional level via gene array, and in cell culture using recultivation assays. Genes differentially expressed in SP cells were evaluated at immunohistochemistry in tissue samples from 486 patients with gastric cancer. The SP cells were smaller and rounder then non-SP cells. SP cells self-renewed in recultivation experiments and differentiated into SP and non-SP cells. Recultivated SP and non-SP cells exhibited distinct phenotypes in culture insofar as cell shape and colony formation. SP cells demonstrated increased levels of the stem cell markers CD133 and Musashi-1. Transcriptional analyses demonstrated that SP cells express genes that encode for stem cell properties including FZD7, HEY1, SMO, and ADAM17. It was observed that ADAM17 and FZD7 are differentially expressed in human gastric cancer, and FZD7-positive cancers are associated with significantly shorter patient survival. In conclusion, human gastric cancer cell lines enclose a phenotypically and genotypically distinct cell population with tumor stem cell features. Phenotypic characteristics of this distinct cell population are also present in gastric cancer tissue, and correlate with patient survival.
| S-EPMC3078445 | biostudies-literature
Project description:Prolonged estrogen exposure is believed to be the major cause of endometrial cancer. As possible markers of estrogen exposure, various menstrual and reproductive features, e.g., ages at menarche and menopause, are found to be associated with endometrial cancer risk. In order to assess their combined effects on endometrial cancer, we created the total number of menstrual cycles (TNMC) that a woman experienced during her life or up to the time of study and two genetic risk scores, GRS1 for age at menarche and GRS2 for age at menopause. Comparing 482 endometrial cancer patients with 571 population controls, we found TNMC was associated with endometrial cancer risk and that the association remained statistically significant after adjustment for obesity and other potential confounders. Risk increased by about 2.5% for every additional 10 menstrual-cycles. The study also showed that high GRS1 was associated with increased risk. This relationship, however, was attenuated after adjustment for obesity. Our study further indicated women with high TNMC and GRS1 had twice the risk of endometrial cancer compared to those low in both indices. Our results provided additional support to the involvement of estrogen exposure in endometrial cancer risk with regard to genetic background and lifestyle features.
| S-EPMC4620445 | biostudies-literature
Project description:BackgroundIn Asherman's syndrome (AS), intrauterine scarring and fibrotic adhesions lead to menstrual disorders, pregnancy loss, or infertility. A few clinical trials have piloted cell therapy to overcome AS. Understanding the role of the stromal compartment in endometrial regeneration remains poorly understood. We hypothesize that endometrial stromal cells (eSCs) represent a relevant cell population to establish novel cell-based therapeutics for endometrial disorders. The aim of this study was to characterize eSCs and evaluate their immune-cell interactions.MethodseSCs were isolated from healthy donors, during the proliferative stage of the menstrual cycle. Cells were characterized for expression of mesenchymal stromal cell (MSC) markers and assessed for their tumorigenic potential. eSCs were co-cultured with interferon γ and tumor necrosis factor α, and cell surface expression of their respective receptors and human leukocyte antigen (HLA) I and II determined by flow cytometry. Secreted levels of key immunomodulatory factors were established. eSCs were cultured with activated peripheral blood mononuclear cells, and T cell differentiation and proliferation determined.ResultseSCs demonstrated an MSC surface phenotype and exhibited multipotency. Expanded eSCs retained chromosomal stability and demonstrated no tumorigenicity. Upon stimulation, eSCs licensed to an anti-inflammatory phenotype with upregulated secretion of immunomodulatory factors. Stimulated eSCs did not express HLA class II. eSCs suppressed the proliferation and activation of CD4+ T cells, with the eSC secretome further downregulating central memory T cells and upregulating effector memory (EM) cells.ConclusionsDifferential responsiveness to inflammation by eSCs, compared to other MSC sources, demonstrates the need to understand the specific functional effects of individual stromal cell sources. A lack of HLA class II and triggering of EM T cell differentiation strongly links to innate in vivo roles of eSCs in tissue repair and immune tolerance during pregnancy. We conclude that eSCs may be an ideal cell therapy candidate for endometrial disorders.
| S-EPMC6945659 | biostudies-literature
Project description:BackgroundThe human endometrium undergoes cyclical regeneration throughout a woman's reproductive life. Ectopic implantation of endometrial cells through retrograde menstruation gives rise to endometriotic lesions which affect approximately 10% of reproductive-aged women. The high regenerative capacity of the human endometrium at eutopic and ectopic sites suggests the existence of stem/progenitor cells and a unique angiogenic system. The objective of this study was to isolate and characterize putative endometrial stem/progenitor cells and to address how they might be involved in the physiology of endometrium.Methodology/principal findingsWe found that approximately 2% of the total cells obtained from human endometrium displayed a side population (SP) phenotype, as determined by flow cytometric analysis of Hoechst-stained cells. The endometrial SP (ESP) cells exhibited preferential expression of several endothelial cell markers compared to endometrial main population (EMP) cells. A medium specific for endothelial cell culture enabled ESP cells to proliferate and differentiate into various types of endometrial cells, including glandular epithelial, stromal and endothelial cells in vitro, whereas in the same medium, EMP cells differentiated only into stromal cells. Furthermore, ESP cells, but not EMP cells, reconstituted organized endometrial tissue with well-delineated glandular structures when transplanted under the kidney capsule of severely immunodeficient mice. Notably, ESP cells generated endothelial cells that migrated into the mouse kidney parenchyma and formed mature blood vessels. This potential for in vivo angiogenesis and endometrial cell regeneration was more prominent in the ESP fraction than in the EMP fraction, as the latter mainly gave rise to stromal cells in vivo.Conclusions/significanceThese results indicate that putative endometrial stem cells are highly enriched in the ESP cells. These unique characteristics suggest that ESP cells might drive physiological endometrial regeneration and be involved in the pathogenesis of endometriosis.
| S-EPMC2860997 | biostudies-literature
Project description:BackgroundEndometrial stem/progenitor cells contribute to the cyclical regeneration of human endometrium throughout a woman's reproductive life. Although the candidate cell populations have been extensively studied, no consensus exists regarding which endometrial population represents the stem/progenitor cell fraction in terms of in vivo stem cell activity. We have previously reported that human endometrial side population cells (ESP), but not endometrial main population cells (EMP), exhibit stem cell-like properties, including in vivo reconstitution of endometrium-like tissues when xenotransplanted into immunodeficient mice. The reconstitution efficiency, however, was low presumably because ESP cells alone could not provide a sufficient microenvironment (niche) to support their stem cell activity. The objective of this study was to establish a novel in vivo endometrial stem cell assay employing cell tracking and tissue reconstitution systems and to examine the stem cell properties of ESP through use of this assay.Methodology/principal findingsESP and EMP cells isolated from whole endometrial cells were infected with lentivirus to express tandem Tomato (TdTom), a red fluorescent protein. They were mixed with unlabeled whole endometrial cells and then transplanted under the kidney capsule of ovariectomized immunodeficient mice. These mice were treated with estradiol and progesterone for eight weeks and nephrectomized. All of the grafts reconstituted endometrium-like tissues under the kidney capsules. Immunofluorescence revealed that TdTom-positive cells were significantly more abundant in the glandular, stromal, and endothelial cells of the reconstituted endometrium in mice transplanted with TdTom-labeled ESP cells than those with TdTom-labeled EMP cells.Conclusions/significanceWe have established a novel in vivo endometrial stem cell assay in which multi-potential differentiation can be identified through cell tracking during in vivo endometrial tissue reconstitution. Using this assay, we demonstrated that ESP cells differentiated into multiple endometrial lineages in the niche provided by whole endometrial cells, indicating that ESP cells are genuine endometrial stem/progenitor cells.
| S-EPMC3514174 | biostudies-literature
Project description:Endometrial cancer is the most common gynecologic cancer with high heterogeneity. However, there are limited treatment options for advanced endometrial carcinoma. In recent years, immunotherapy has broadly been used for the treatment of various cancers. However, the efficacy of immunotherapy against endometrial cancer is limited. The tumor microenvironment, including mesenchymal stem cells (MSCs), may contribute to tumor progression through cancer cells themselves and through cells of the immune system. We successfully isolated endometrial cancer-derived MSCs (EmCaMSCs) from patients and found that the population of MSCs in tumor tissues was approximately 1%-5%. The population of MSCs correlated with the stage of the disease. EmCaMSCs expressed MSC markers and exhibited trilineage differentiation ability. The programmed death ligands PD-L1 and PD-L2 were highly expressed in EmCaMSCs; their expression could be further enhanced by tumor necrosis factor-α and interferon-γ. When cocultured with peripheral blood mononuclear cells (PBMCs), anti-CD3, and anti-CD28, EmCaMSCs inhibited the proliferation of PBMCs, which were partially rescued by treatment with anti-PD-L1 antibodies. From the profile of conditioned medium of EmCaMSCs, we discovered that interleukin (IL)-8 and insulin-like growth factor-binding protein 6 could also rescue the proliferation of PBMCs. Furthermore, EmCaMSCs cocultured with IL-2-induced PBMCs exhibited decreased expression of CD56, CD4, and CD8 and showed decreased cytotoxicity toward K562 cells and endometrial cancer cells. Overall, EmCaMSCs were isolated successfully from endometrial cancer tissues and exhibited immunosuppressive effects and may be targeted for the treatment of endometrial cancer by anti-cytokine and immune checkpoint inhibitors. The percentage of MSCs in tumor stroma might predict the prognosis of endometrial cancer.
| S-EPMC9210080 | biostudies-literature