Dataset Information

Single cell RNA sequencing of intestinal fibroblast-crypt co-cultures in organoid growth media with/without Ptger4/EP4 inhibitor (Drop-seq)

ABSTRACT: Mouse small intestinal crypts were co-cultured with primary intestinal fibroblasts in Organoid Growth Media (OGM) with/without inhibitor for the prostaglandin E2 receptor Ptger4 (EP4), labeled respectively as “Ptger4-OFF” and “Ptger4-ON” conditions. Crypts co-cultured with fibroblasts in Ptger4-ON conditions (OGM containing DMSO vehicle control) acquired a spheroid morphology. Crypts co-cultured with fibroblasts in Ptger4-OFF conditions (OGM supplemented with Ptger4 inhibitor) developed into budding organoids. Fibroblasts were isolated from the small intestine of wild type mice. For the co-cultures 2 x 10e4 fibroblasts were seeded in 48-well plates overnight. Freshly isolated crypts (n = 500) were suspended in 1:1 Matrigel (Corning, 356231) and IntestiCult Organoid Growth Medium (Stem Cell Technologies, 06005) and added as an overlay on the fibroblasts. Crypts/fibroblasts were co-cultured with IntestiCult Organoid Growth Medium for 4 days. The ONO-AE3-208 Ptger4 (EP4) inhibitor (Cayman, 14522) dissolved in DMSO was added to the co-cultureson days 0 and 2 at a final concentration of 10 uM. DMSO was used as a vehicle control for the untreated co-cultures. On day 4, one pool of six Ptger4-ON co-cultures and one pool of six Ptger4-OFF co-cultures were subjected to the Drop-seq protocol.

ORGANISM(S): Mus musculus

PROVIDER: GSE142420 | GEO | 2019/12/21

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Recognition of foreign antigens by B cell receptor (BCR) on mature B cells leads to their clonal expansion, which is critically important for the effective host defence of the organism. However, excessive antigenic responses or reaction of B cells against bodyâs own components frequently lead to diverse immune diseases, such as B-cell lymphoma or autoimmunity, that often affect humans. Identification of genes that restrain uncontrolled proliferation of B cells is therefore an important goal towards understanding the origin of such diseases. Here we identify Ptger4 as a negative feedback regulator of B-cell proliferation in response to BCR triggering, and show that its encoded EP4 receptor is a principal molecule conveying the growth-suppressive effect of prostaglandin E2 (PGE2). In controlled in vitro assays, Ptger4-/- B cells showed augmented proliferative response and increased expression of activating genes upon BCR stimulation. Stable knock-down of Ptger4 in B-cell lymphoma markedly accelerated tumour spread in mice, while Ptger4 overexpression yielded significant protection. Lack of Ptger4 rendered mouse B cells completely resistant to proliferation arrest signalled by PGE2, and we find by transcriptional profiling that intrinsic activity of Ptger4 and PGE2-EP4 signalling target a similar set of activating genes. We further show that Ptger4 inhibits mouse B-cell activation in vivo and find it significantly downregulated in human B-cell lymphoma. Our results demonstrate that Ptger4 functions in B cells as a candidate tumour suppressor whose activity is regulated by the presence of PGE2 in the microenvironment. These findings suggest that targeting EP4 receptor for prostaglandin may present a novel strategy for treatment of B-cell diseases. Keywords: time course, cell type comparison, compound treatment design, microarray experiment record B cells were extracted from spleen of Ptger4+/+ and Ptger4-/- mice, and incubated in vitro for the indicated period with or without anti-IgM (Fab')2 antibody fragments and co-treated with or without PGE2. Total RNA was extracted, amplified, labelled with Cy3 or Cy5, and hybridized to mouse 24k oligo-arrays using a dye-swap strategy.

Project description:Fibrotic interstitial lung disease (ILD) are lung disorders characterized by the accumulation of extracellular matrix, ultimately resulting in the destruction of the pulmonary scaffold. Continuous pro-fibrotic signaling perpetuates the remodeling process, specifically targeting the epithelial cell compartment, thereby destroying the gas exchange area. Studies that address this detrimental crosstalk between lung epithelial cells and fibroblasts are key to understanding ILD. With the aim of identifying functionally relevant targets that drive mesenchymal-epithelial crosstalk and their potential as new avenues to therapeutic strategies, we developed an organoid co-culture system based on human induced pluripotent stem cell-derived alveolar epithelial type 2 cells and lung fibroblasts from ILD patients as well as IMR-90 controls. While organoid formation capacity and organoid size was comparable in the presence of ILD or control lung fibroblasts, metabolic activity was significantly increased in ILD co-cultures. Alveolar organoids cultured with ILD fibroblasts further demonstrated reduced stem cell function supported by reduced Surfactant Protein C gene expression together with an aberrant basaloid-prone differentiation program indicated by elevated Cadherin 2, Bone Morphogenic Protein 4 and Vimentin transcription. In order to identify key mediators of the misguided mesenchymal-to-epithelial crosstalk with a focus on disease-relevant inflammatory processes, we used secretome mass spectrometry to identify key signals secreted by end stage ILD lung fibroblasts. Over 2000 proteins were detected in a single-shot experiment with 47 differentially upregulated proteins when comparing ILD and non-chronic lung disease control fibroblasts. The secretome profile was dominated by chemokines of the C-X-C motif family, including CXCL1, -3, and -8, all interfering with (epithelial) growth factor signaling orchestrated by Interleukin 11 (IL11), steering fibrogenic cell-cell communication, and proteins regulating extracellular matrix remodeling including epithelial-to-mesenchymal transition. When in turn treating 3D monocultures of iAT2s with IL11 we recapitulated the co-culture results obtained with primary ILD fibroblasts including changes in metabolic activity as well as organoid formation capacity and size. In summary, our analysis identified mesenchyme-derived mediators likely contributing to the disease-perpetuating mesenchymal-to-epithelial crosstalk in ILD by using sophisticated alveolar organoid co-cultures indicating the importance of cytokine-driven aberrant epithelial differentiation and confirmed IL11 as a key player in ILD using an unbiased approach.

Dataset Information

Single cell RNA sequencing of intestinal fibroblast-crypt co-cultures in organoid growth media with/without Ptger4/EP4 inhibitor (Drop-seq)

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets