Dataset Information

Organoid co-culture model of the human endometrium in a fully synthetic extracellular matrix enables the study of epithelial-stromal crosstalk.

ABSTRACT:

Background

The human endometrium undergoes recurring cycles of growth, differentiation, and breakdown in response to sex hormones. Dysregulation of epithelial-stromal communication during hormone-mediated signaling may be linked to myriad gynecological disorders for which treatments remain inadequate. Here, we describe a completely defined, synthetic extracellular matrix that enables co-culture of human endometrial epithelial and stromal cells in a manner that captures healthy and disease states across a simulated menstrual cycle.

Methods

We parsed cycle-dependent endometrial integrin expression and matrix composition to define candidate cell-matrix interaction cues for inclusion in a polyethylene glycol (PEG)-based hydrogel crosslinked with matrix metalloproteinase-labile peptides. We semi-empirically screened a parameter space of biophysical and molecular features representative of the endometrium to define compositions suitable for hormone-driven expansion and differentiation of epithelial organoids, stromal cells, and co-cultures of the two cell types.

Findings

Each cell type exhibited characteristic morphological and molecular responses to hormone changes when co-encapsulated in hydrogels tuned to a stiffness regime similar to the native tissue and functionalized with a collagen-derived adhesion peptide (GFOGER) and a fibronectin-derived peptide (PHSRN-K-RGD). Analysis of cell-cell crosstalk during interleukin 1B (IL1B)-induced inflammation revealed dysregulation of epithelial proliferation mediated by stromal cells.

Conclusions

Altogether, we demonstrate the development of a fully synthetic matrix to sustain the dynamic changes of the endometrial microenvironment and support its applications to understand menstrual health and endometriotic diseases.

Funding

This work was supported by The John and Karine Begg Foundation, the Manton Foundation, and NIH U01 (EB029132).

SUBMITTER: Gnecco JS

PROVIDER: S-EPMC10878405 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Organoid co-culture model of the human endometrium in a fully synthetic extracellular matrix enables the study of epithelial-stromal crosstalk.

Gnecco Juan S JS Brown Alexander A Buttrey Kira K Ives Clara C Goods Brittany A BA Baugh Lauren L Hernandez-Gordillo Victor V Loring Megan M Isaacson Keith B KB Griffith Linda G LG

Med (New York, N.Y.) 20230801 8

<h4>Background</h4>The human endometrium undergoes recurring cycles of growth, differentiation, and breakdown in response to sex hormones. Dysregulation of epithelial-stromal communication during hormone-mediated signaling may be linked to myriad gynecological disorders for which treatments remain inadequate. Here, we describe a completely defined, synthetic extracellular matrix that enables co-culture of human endometrial epithelial and stromal cells in a manner that captures healthy and diseas ...[more]

PMID: 37572651

Similar Datasets

Project description:Porcine (Sus scrofa domesticus) small intestinal mucosal layers from the ‘Pietrain’ breed were used. To harvest the intestine, one end was cut in situ and the internal tube was pulled out leaving behind the external layer and mesentery. The retrieved mucosal tissue is then extensively cleaned with deionized water (dH2O), opened longitudinally, cut into 1 cm pieces and placed in dH2O to begin the first step of decellularization. The 1cm pieces of intestine were placed in dH2O overnight at 4°C and then in 4% sodium deoxycholate for 4hr at room temperature (RT). This was followed by a washing step in PBS (phosphate buffer saline) for 30min at RT and then 2000kU DNase-I in 1M NaCl for 3hr at RT. The tissue was then placed in milliQ water and washed for 3 days at 4C, changing the water daily. A magnetic stirrer was used throughout the decellularization process. The decellularized porcine intestine was freeze dried overnight or until completely dry, milled into a thin powder using a mini mill, gamma irradiated (17 kGy for 10h) and stored at -20C until further use. The lyophilized ECM powder was split into 3 biological replicates, each processed independently and analyzed in triplicate by LC-MS/MS. The powder was resuspended in lysis buffer and two spike-in proteins (each at 0.5 µg/100 µg of protein powder) were added: carnitine monooxygenase oxygenase subunit (cntA, D0C9N6) from Acinetobacter baumannii, and CTP synthase (CTPsyn, Q9VUL1) from Drosophila melanogaster. Protein extraction was performed by heating at 90°C for 10 min, and centrifuging at maximum velocity for 10 min at 4°C. ECM-derived proteins were reduced in 0.1 M dithiothreitol (DTT) at 95°C for 5 min, dissolved in 8 M urea solution after cooling down to room temperature, alkylated with 55 mM iodoacetamide for 30 min at 25°C in the dark. Alkylated proteins were purified using Microcon YM-10 filter unit for 8 times at 14000g for 40min followed by trypsin digestion for 16 h at 37°C. pH was adjusted to 3 by addition of formic acid. Peptides were desalted by C-18 column and dried into powder and were then re-suspended in 30 µl 0.1% acetic acid for the following mass spectrometry analysis. Protein identification by liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed using Thermo Fusion Mass Spectrometer with Thermo Easy-nLC1000 Liquid Chromatography. 130 min of LC-MS gradients were performed by increasing organic proportion. The first level of MS was detected by Orbitrap with parameter of Resolution at 120K, Scan Rang at 300-1800 m/z, Mass Tolerance at 10 ppm. The second level of MS was isolated by Quadrupole, activated by HCD and detected by Orbitrap. The Orbitrap Resolution for the second level of MS was 30K. Peak files .mzXML were obtained by RawConverter starting from .raw files. Starting from peak files, the mass spectrometry–derived data were searched against a human protein database (Uniprot Homo sapiens reference proteome, UP000005640) by MaxQuant v. 1.6.7.0. Oxidation of methionine residues and acetyl of protein N-term were set as variable modifications. Carbamidomethyl on cysteine was set as fixed modification. Peptide-spectrum matches (PSMs) were adjusted to a 1% and then assembled further to a final protein-level false discovery rate (FDR) of 1%.

Project description:Mucosal barrier tissues, comprising a layer of tightly-bonded epithelial cells in intimate molecular communication with an underlying matrix-rich stroma containing fibroblasts and immune cells, are prominent targets for drugs against infection, chronic inflammation, and other disease processes. Although human in vitro models of such barriers are needed for mechanistic studies and drug development, differences in extracellular matrix (ECM) needs of epithelial and stromal cells hinder efforts to create such models. Here, using the endometrium as an example mucosal barrier, we describe a synthetic, modular ECM hydrogel suitable for 3D functional co-culture, featuring components that can be remodeled by cells and that respond dynamically to sequester local cell-secreted ECM characteristic of each cell type. The synthetic hydrogel combines peptides with off-the-shelf reagents and is thus accessible to cell biology labs. Specifically, we first identified a single peptide as suitable for initial attachment of both endometrial epithelial and stromal cells using a 2D semi-empirical screen. Then, using a co-culture system of epithelial cells cultured on top of gel-encapsulated stromal cells, we show that inclusion of ECM-binding peptides in the hydrogel, along with the integrin-binding peptide, leads to enhanced accumulation of basement membrane beneath the epithelial layer and more fibrillar collagen matrix assembly by stromal cells over two weeks in culture. Importantly, endometrial co-cultures composed of either cell lines or primary cells displayed hormone-mediated differentiation as assessed by morphological changes and secretory protein production. A multiplex analysis of apical cytokine and growth factor secretion comparing cell lines and primary cells revealed strikingly different patterns, underscoring the importance of using primary cell models in analysis of cell-cell communication networks. In summary, we define a "one-size-fits-all" synthetic ECM that enables long-term, physiologically responsive co-cultures of epithelial and stromal cells in a mucosal barrier format.

Dataset Information

Organoid co-culture model of the human endometrium in a fully synthetic extracellular matrix enables the study of epithelial-stromal crosstalk.

Background

Methods

Findings

Conclusions

Funding

Publications

Organoid co-culture model of the human endometrium in a fully synthetic extracellular matrix enables the study of epithelial-stromal crosstalk.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets