Project description:Analysis of human alveolar epithelial cell signatures at gene expression level. The aim of the study was to identify candidate genes that are induced in human alveolar epithelial type II cells on collagen-coated dishes. Results provide important information about changes hAT2 cells undergo in the transformation to AT1-like cells. Total RNA obtained from human alveolar epithelial cells grown on collagen- or matrigel-coated dishes for 12, 24, 48 and 72 hr.
Project description:Analysis of human alveolar epithelial cell signatures at gene expression level. The aim of the study was to identify candidate genes that are induced in human alveolar epithelial type II cells on collagen-coated dishes. Results provide important information about changes hAT2 cells undergo in the transformation to AT1-like cells.
Project description:Myeloid Angiogenic Cells (MACs) were infected with the intracellular, bacterial pathogen Bartonella henselae (B.h.). Infected cells were seeded onto Matrigel coated plates. While uninfected cells showed no phenotypic changes and died over time, infected cells showed strong phenotypic changes and developed into complex 2D chord networks over the course of long term culture (eg 49d). To examine the changes in gene expression associated with the development of the B.h.dependent chord formation phenotype, RNA was isolated from MACs shortly after isolation (d4) and from cells of the chord structures (+B.h. Matrigel). As primary endothelial cells are also know to form chord networks when cultured on Matrigel, a sample of human umbilical vein endothelial cells (HUVECs) cultured on Matrigel for 12hr was also included in the analysis as a control. myeloid angiogenic cells (MACs) from three donors were compared d4 after isolation with MACs infected with Bartonella henselae and cultured on Matrigel coated plates for up to 49 days, 1 sample from human umbilical cord vein endothelial cells (HUVECs) cultured for 12hr on Matrigel coated plates were also included as a control.
Project description:To elucidate potential mechanisms how EPB41L5 modulates integrin adhesion complex maturation, we performed quantitative SILAC-based integrin adhesome proteomics. For MS analysis of the podocyte adhesome, EPB41L5 or Luciferase (as negative control) was transient expressed in SILAC labeled podocytes by nucleofection (Amaxa Nucleofector, Lonza, Switzerland). Podocytes were seeded on cell culture dishes for 24 hours. Isolation of integrin adhesion complexes from podocytes was performed prior LC-MSMS analysis.In a second set of experiments adhesion complexes of collagen IV and fibronectin coated dishes were purified and analysed by LC-MSMS.
Project description:To address the need for human alveolar epithelial cell (AEC)-derived lines to more suitably model distal lung diseases, we have generated and characterized novel immortalized cell lines derived from human AECs. We used a combination of the ROCK inhibitor, Y-27632, and lentiviral transduction of SV40 Large T antigen of previously cryopreserved isolated human alveolar epithelial type 2 (AT2) cells to generate immortalized AECs. These AEC lines proliferate well on standard tissue culture dishes forming an epithelial monolayer and express lung progenitor markers SOX9 and SOX2. When grown in 3D culture with lung fibroblasts, the cells form NKX2-1+ organoids expressing more mature alveolar lung markers, AQP5 and GPRC5A. Single cell RNA-sequencing of one AEC line comparing cells in 2D versus 3D revealed increased cellular heterogeneity and an induction of cytokine and lipoprotein signaling in 3D culture, reflecting interactions with the microenvironment during organoid formation. Taken together, these data show our novel progenitor-like AEC lines retain a genetic and structural memory of their alveolar cell lineage despite long-term expansion, providing a valuable new system to model the distal lung in vitro.
Project description:To address the need for human alveolar epithelial cell (AEC)-derived lines to more suitably model distal lung diseases, we have generated and characterized novel immortalized cell lines derived from human AECs. We used a combination of the ROCK inhibitor, Y-27632, and lentiviral transduction of SV40 Large T antigen of previously cryopreserved isolated human alveolar epithelial type 2 (AT2) cells to generate immortalized AECs. These AEC lines proliferate well on standard tissue culture dishes forming an epithelial monolayer and express lung progenitor markers SOX9 and SOX2. When grown in 3D culture with lung fibroblasts, the cells form NKX2-1+ organoids expressing more mature alveolar lung markers, AQP5 and GPRC5A. Single cell RNA-sequencing of one AEC line comparing cells in 2D versus 3D revealed increased cellular heterogeneity and an induction of cytokine and lipoprotein signaling in 3D culture, reflecting interactions with the microenvironment during organoid formation. Taken together, these data show our novel progenitor-like AEC lines retain a genetic and structural memory of their alveolar cell lineage despite long-term expansion, providing a valuable new system to model the distal lung in vitro.
Project description:We aimed to analyse the effect of different extra-cellular matrices on the growth of small intestinal organoids. Small intestinal crypts of wildtype mice were harvested and grown under standard Matrigel organoid conditions. After establishment of organoids (passaged 1-2), organoids were grown in Matrigel, on collagen or in a drop of collagen. Growth in a droplet of collagen requires addition of Wnt3a, therefore all samples are either provided with standard culture conditions (ENR) or with ENR+50%Wnt3a-CM (WENR). Samples were grown in specified matrix for at least 1-2 passages before RNA was purified.
Project description:SVG-A cells are immortalized fetal astrocytes that respond to Notch signaling. We characterized the transcriptional response to Notch activation in this cell line by performing RNA-seq on cells grown on tissue culture dishes coated with JAGGED1, a Notch ligand. Differential gene expression analysis shows induction of both canonical Notch responsive genes, as well as cell-type specific Notch responsive genes.
Project description:Myeloid Angiogenic Cells (MACs) were infected with the intracellular, bacterial pathogen Bartonella henselae (B.h.). Infected cells were seeded onto Matrigel coated plates. While uninfected cells showed no phenotypic changes and died over time, infected cells showed strong phenotypic changes and developed into complex 2D chord networks over the course of long term culture (eg 49d). To examine the changes in gene expression associated with the development of the B.h.dependent chord formation phenotype, RNA was isolated from MACs shortly after isolation (d4) and from cells of the chord structures (+B.h. Matrigel). As primary endothelial cells are also know to form chord networks when cultured on Matrigel, a sample of human umbilical vein endothelial cells (HUVECs) cultured on Matrigel for 12hr was also included in the analysis as a control.
Project description:RNA-seq data from human mammary epithelial MCF-10A cells expressing the coding sequence of SRSF2, SRSF4, SRSF6, SRSF9, TRA2B, or a control empty vector plasmid grown for 8 days in 3D culture in matrigel