Gene expression profiling of epithelial cells from human cervix cultured in Organ Chip and transwells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE231016 | GEO | 2023/12/12
REPOSITORIES: GEO
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Project description:Modulation of mucus production by the human endo- and ecto-cervical epithelium by steroid hormones and associated interactions with commensal microbiome play a central role in the physiology and pathophysiology of the female reproductive tract. However, most of our knowledge about these interactions is based on results from animal studies or in vitro models that fail to faithfully mimic the mucosal environment of the human cervix. Here we describe microfluidic organ-on-a-chip (Organ Chip) models of the human cervical mucosa that recreate the cervical epithelial-stromal interface with a functional epithelial barrier and produce abundant mucus that has compositional, biophysical, and hormone-responsive properties similar to the living cervix. Application of continuous fluid flow to chips lined primary human cervical epithelial cells from a commercial source that contained a mixture of primary human ecto- and endo-cervical epithelial cells promoted preferential expression of the ecto-cervical phenotype, whereas use of periodic flow including periods of stasis induced endo-cervical specialization. When the periodic flow Cervix Chips were co-cultured with living Lactobacillus crispatus and Gardnerella vaginalis bacterial communities to respectively mimic the effects of human host interactions with optimal (healthy) or non-optimal (dysbiotic) microbiome associated with an ascending infection in the female reproductive tract, significant differences in tissue innate immune responses, barrier function, cell viability and protein profile, and mucus composition were detected reminiscent of those observed in vivo. Thus, these Organ Chip models of human cervix provide a physiologically relevant experimental in vitro model to study cervical mucus physiology and its role in human host-microbiome interactions as well as a potential preclinical testbed for development of therapeutic interventions to enhance women's health.
2023-12-11 | MSV000093636 | MassIVE
Project description:Modulation of mucus production by the human ecto- and endo-cervical epithelium by steroid hormones and associated interactions with commensal microbiome play a central role in the physiology and pathophysiology of the female reproductive tract. However, most of our knowledge about these interactions is based on results from animal studies or in vitro models that fail to faithfully mimic the mucosal environment of the human cervix. Here we describe microfluidic organ-on-a-chip (Organ Chip) models of the human cervical mucosa that recreate the cervical epithelial-stromal interface with a functional epithelial barrier and produce abundant mucus that has compositional, biophysical, and hormone-responsive properties similar to the living cervix. Use of continuous fluid flow promoted ecto-cervical differentiation, whereas use of periodic flow including periods of stasis stimulated endo-cervical specialization. Similar results with minor differences were obtained using epithelial cells isolated from three donors each from a different ethnic background (African American, Hispanic, and Caucasian). When the endo-Cervix Chips were co-cultured with living Lactobacillus crispatus and Gardnerella vaginalis bacterial communities to respectively mimic the effects of human host interactions with optimal (healthy) or non-optimal (dysbiotic) microbiome, significant differences in tissue innate immune responses, barrier function, cell viability, and mucus composition were detected reminiscent of those observed in vivo. Thus, human Cervix Chips provide a physiologically relevant experimental in vitro model to study cervical mucus physiology and its role in human host-microbiome interactions as well as a potential preclinical testbed for development of therapeutic interventions to enhance women's health.
2023-04-26 | MSV000091806 | MassIVE
Project description:The transition zones (TZs) of the squamous and columnar epithelium constitute hotspots for the emergence of cancers, often preceded by metaplasia, where one epithelial type is replaced by cells of another type. Yet, it remains uncertain how the spatial organization of the epithelia is maintained and how the TZ niche is remodeled during metaplasia. Here, we used single-cell RNA-sequencing to characterize subpopulations of the epithelium as well as the underlying stromal compartment of endo and ectocervix, encompassing the TZ. Mouse lineage tracing, organoid culture and smRNA-ISH revealed that the two epithelia derive from two separate cervix-resident lineage-specific stem cell populations that are regulated by opposing WNT signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodeling and increased expression of WNT signaling inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Thus, homeostasis at the TZ results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.
2020-12-26 | GSE128987 | GEO
Project description:Cervical cancer is a common gynecological malignancy, often caused by high-risk human papillomavirus. There is a paucity of human-derived culture systems to study cervical epithelium and the cancers derived thereof. Here, we describe a long-term culturing protocol for ecto- and endocervical epithelia, which generates 3D organoids that stably recapitulate the two tissues of origin. In the example of successful HSV-1 infection, we show that the organoid-based cervical models serve as promising platforms for studying sexually transmitted pathogens. Starting from Pap-brush material, a small biobank of patient-derived tumoroids was established that retained the causative HPV genomes. One of these uniquely carried the poorly characterized HPV30 subtype, implying a potential role in carcinogenesis. The tumoroids displayed differential responses to common chemotherapeutics and grew as xenografts in mice. This study describes an experimental platform for cervical (cancer) research and for future personalized medicine approaches. Purpose: Transcriptome analysis was performed on organoids/tissues derived from human ecto- and endocervix as well as associated malignancies to characterize the new organoid-based platform. Methods: Ectocervical organoids and tumoroid lines were cultured in M7 media, whereas endocervical organoids in M7+4 media prior harvesting for RNA extraction (see paper for details). Additionally, RNA was extracted from healthy ecto-and endocervical tissue biopsies as well as from primary cells collected via Pap brush method from cervical cancer patients. The libraries were prepared by the Utrecht Sequencing Facility (USEQ) based on polyA enrichment. Sequencing was performed on an Illumina NextSeq500 by using 75-bp single-end or paired-end sequencing. Reads were aligned to the human reference genome (GRCh37) using Burrows-Wheeler Aligner (BWA) (v0.5.9). Viral sequences of different HPV subtypes were detected in the RNA-seq data of the different tumoroid lines and tissues by using VirusSeq-CLI, a wrapper around the VirusSeq pipeline. Results: Ectocervical and endocervical organoids display distinct transcriptomic profiles that can be maintained in cultures for long term. Similarily, the the gene expression profile of tumor organoids clusters apart from healthy counterparts and shows evident tumor-like characteristics. Tumor organoids show expression of causative HPV oncogenes.
2021-03-05 | GSE168244 | GEO
Project description:We report transcriptomic analysis of CD4 T cells isolated from vaginal, ectocervical, and endocervical tissues from 4 individuals.
2021-04-28 | GSE163260 | GEO
Project description:The cellular origin of cervical cancers remains unclear. Revealing molecular details of transformation in this tissue has been hampered by the lack of culture systems, resembling the in vivo cervical architecture. Here we established a long-term in vitro 3D cervical organoid model derived from stem cells of human or mouse cervical tissue which recapitulates the in vivo stratified ectocervical and columnar endocervical epithelium. Stratified and columnar cervical epithelia arise from two discrete unipotent stem cell populations of the endocervix. Unique stem cell signatures reveal a dependency on intrinsic Notch and Wnt microenvironmental signals. The genetic signatures of KRT5+ stratified vs KRT7+ columnar cervical cells establish discrete groups of cervical cancer of the squamous and adenocarcinoma types, respectively. Cervical tissue morphology is guided by the interplay of two discrete unipotent cervical stem cell populations and the spatio-temporal distribution of signals from the stroma.
2019-09-01 | GSE87075 | GEO
Project description:The cellular origin of cervical cancers remains unclear. Revealing molecular details of transformation in this tissue has been hampered by the lack of culture systems, resembling the in vivo cervical architecture. Here we established a long-term in vitro 3D cervical organoid model derived from stem cells of human or mouse cervical tissue which recapitulates the in vivo stratified ectocervical and columnar endocervical epithelium. Stratified and columnar cervical epithelia arise from two discrete unipotent stem cell populations of the endocervix. Unique stem cell signatures reveal a dependency on intrinsic Notch and Wnt microenvironmental signals. The genetic signatures of KRT5+ stratified vs KRT7+ columnar cervical cells establish discrete groups of cervical cancer of the squamous and adenocarcinoma types, respectively. Cervical tissue morphology is guided by the interplay of two discrete unipotent cervical stem cell populations and the spatio-temporal distribution of signals from the stroma.
2019-09-01 | GSE87074 | GEO
Project description:Genome wide DNA methylation profiling of normal and squamous carcinoma of the cervix. The Illumina Infinium 27k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,000 CpGs in frozen cervical samples. Samples included 4 normal ectocervices (Ecto 1 to 4) and 5 squamous cell carcinoma of the cervix (SCC 1 to 5). In order to identify epigenetic biomarkers for early cervix cancer diagnosis, we performed a methylation screening using Illumina Infinium 27k Human DNA methylation Beadchip v1.2 of stem cell marker promoters during cervical carcinogenesis and demonstrated a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in squamous cell carcinoma (SCC) in comparison with normal ectocervix. Direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples showed that UTF1 promoter methylation increases with lesion severity and is associated with higher expression. By RT-PCR, Western Blot and immunofluorescence, we demonstrated that UTF1 mRNA and protein are expressed in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Methyl-Specific PCR experiments revealed that the inhibition of DNA methyltransferase by 5-aza-2’-deoxycytidine is associated with decreased UTF1 gene methylation and expression. These findings provide evidence that UTF1 promoter methylation profile might be a useful biomarker for cervix cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms involved in the regulation of its expression. Bisulfite converted DNA from the 9 samples were hybridised to the Illumina Infinium 27k Human Methylation Beadchip v1.2
2012-03-21 | E-GEOD-36637 | biostudies-arrayexpress
Project description:A better understanding of the consequences of recurrent (epi)genetic alterations that occur during cervical carcinogenesis is essential in the search for novel biomarkers. In this study we determined genome-wide expression profiles of 10 squamous cell carcinomas (SCCs), 5 adenocarcinomas (AdCAs) and 6 normal epithelial samples. Expression patterns were subsequently combined with previously determined chromosomal profiles in the same carcinomas. Differential gene expression analysis identified 76 genes with altered expression in carcinomas compared to normal epithelium. Microarray results for a subset of these genes were validated by real-time RT-PCR. Among the differentially expressed genes a relative overrepresentation of genes located at chromosome 3q, one of the most frequently gained areas in SCCs, was observed (false discovery rate (FDR)<0.005). To further investigate the relationship between gene expression and chromosomal alterations 2 statistical approaches were used, i.e. differential gene locus mapping (DIGMAP) and the array CGH expression integration tool (ACE-it). Using these methods we found that increased gene expression was linked to increased gene copy numbers at 1q32.1, 3q13.32-22.3, 3q26.32-27.3, and 20q11.21-13.33, whereas a loss at 11q22.3-25 correlated with recurrent decreased gene expression. Integrated genome-wide chromosomal and transcriptional analysis of cervical carcinomas highlighted 7 genes (i.e. FLJ21291, DTX3L, CCDC14, MCM2, PIK3R4, ATP2C1 and SLC25A36), which were identified by differential gene expression analysis and were located within the chromosomal regions identified by DIGMAP and/or ACE-it as well. Further investigations of these promising marker genes in warranted. Keywords: microarray analysis, array CGH, cervical cancer For microarray mRNA expression analysis 10 SCCs, 5 AdCAs and 6 normal epithelial controls were used. Normal cervical controls consisted of 1 pool of 3 normal cervices distant from tumour, 1 pool of 4 normal ectocervical smears and 1 pool of 5 normal endocervical smears. To expand our number of normal squamous epithelial controls, we included expression profiles of 3 uvulas of non-cancer patients who underwent uvulopalatopharyngoplasty. In addition, we hybridised RNA isolated from 2 different biopsies of the same SCC (SCC12 and SCC13) as a biological replicate. The pool of 4 normal ectocervical smears was hybridised twice as a technical replicate to determine technical variation. Genomic profiling of the same 10 SCCs and 5 AdCAs was done using array CGH (Wilting et al, J Pathol 2006, volume 209, p 220-30).
2010-06-25 | E-GEOD-6473 | biostudies-arrayexpress
Project description:The cervical epithelium undergoes continuous changes in proliferation, differentiation and function that is critical before pregnancy to ensure fertility and during pregnancy to provide a physical and immunoprotective barrier for pregnancy maintenance. Barrier disruption can lead to the ascension of pathogens that elicit inflammatory responses and preterm birth. Here we identify epithelial subtypes in the cervix of nonpregnant, pregnant and laboring mice using single cell- transcriptome and spatial analysis. We identify heterogeneous subpopulations of epithelia displaying spatial and temporal specificity in cell proliferation, turnover and transcriptional programs. Further we identify untimely proliferation and function of epithelial cells in a model of cervical epithelial barrier disruption. Together these data provide insights as to how the cervical epithelium undergoes a continuous remodeling to maintain a dynamically shifting state of homeostasis in pregnancy and labor and provide a framework by which to understand perturbations in epithelial cell health and host-microbe interactions that contribute to risk of premature birth.
2022-05-02 | GSE196529 | GEO