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Integrin-activating Yersinia protein Invasin sustains long-term expansion of primary epithelial cells as 2D organoid sheets [scRNA-Seq]

ABSTRACT: Matrigel®/BME®, a solubilized basement membrane-like preparation, is especially rich in the Extra-Cellular Matrix (ECM) protein laminin-111. This hydrogel supports the formation and long-term growth of epithelial 3D organoids from adult stem cells (ASC)1,2. Laminin111 activates integrin β1 complexes, thus preventing anoikis and driving epithelial polarization. Here, we address if a bacterial integrin-activating protein can mimic Matrigel/BME. Enteropathogenic Yersinia bacteria invade human gut epithelial cells using their outer membrane protein Invasin. This protein binds and activates a diversity of integrin β1 complexes, including the laminin111-specific α6β1 receptor. A recombinant 25 kDa C-terminal integrin-binding fragment of Invasin allowed adhesion of gut epithelial cells, when coated on culture plates. Upon addition of organoid growth factor medium, the epithelial cells grew out in 2D and could be expanded and passaged long-term. Polarization, junction formation and generation of various intestinal cell types (i.e. enterocytes, goblet cells, Paneth cells, and enteroendocrine cells) was stable over time. Sustained expansion of multiple other human-, mouse-, and even snake epithelia was accomplished under comparable conditions. The 2D culturing format holds advantages over the 3D ‘in gel’ format in terms of imaging, accessibility of basal and apical domains and automation for high throughput screening approaches. Invasin represents a fully defined, affordable, versatile, and animal-free complement to Matrigel/BME.

ORGANISM(S): Homo sapiens

PROVIDER: GSE275239 | GEO | 2024/11/29

REPOSITORIES: GEO

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Integrin-activating Yersinia protein Invasin sustains long-term expansion of primary epithelial cells as 2D organoid sheets [RNA-Seq]

Project description:Matrigel®/BME®, a solubilized basement membrane-like preparation, is especially rich in the Extra-Cellular Matrix (ECM) protein laminin-111. This hydrogel supports the formation and long-term growth of epithelial 3D organoids from adult stem cells (ASC)1,2. Laminin111 activates integrin β1 complexes, thus preventing anoikis and driving epithelial polarization. Here, we address if a bacterial integrin-activating protein can mimic Matrigel/BME. Enteropathogenic Yersinia bacteria invade human gut epithelial cells using their outer membrane protein Invasin. This protein binds and activates a diversity of integrin β1 complexes, including the laminin111-specific α6β1 receptor. A recombinant 25 kDa C-terminal integrin-binding fragment of Invasin allowed adhesion of gut epithelial cells, when coated on culture plates. Upon addition of organoid growth factor medium, the epithelial cells grew out in 2D and could be expanded and passaged long-term. Polarization, junction formation and generation of various intestinal cell types (i.e. enterocytes, goblet cells, Paneth cells, and enteroendocrine cells) was stable over time. Sustained expansion of multiple other human-, mouse-, and even snake epithelia was accomplished under comparable conditions. The 2D culturing format holds advantages over the 3D ‘in gel’ format in terms of imaging, accessibility of basal and apical domains and automation for high throughput screening approaches. Invasin represents a fully defined, affordable, versatile, and animal-free complement to Matrigel/BME.

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Integrin-activating Yersinia protein Invasin sustains long-term expansion of primary epithelial cells as 2D organoid sheets [scRNA-Seq]

Project description:Integrin-activating Yersinia protein Invasin sustains long-term expansion of primary epithelial cells as 2D organoid sheets [scRNA-Seq]

| PRJNA1150084 | ENA

Integrin-activating Yersinia protein Invasin sustains long-term expansion of primary epithelial cells as 2D organoid sheets [RNA-Seq]

Project description:Integrin-activating Yersinia protein Invasin sustains long-term expansion of primary epithelial cells as 2D organoid sheets [RNA-Seq]

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Activation of adult human pancreatic ductal and ductal progenitor-like cells requires extracellular matrix protein signaling [scRNA-seq]

Project description:Ductal progenitor-like cells are a subpopulation of ductal cells in the adult human pancreas that have the potential to contribute to regenerative medicine. However, the microenvironmental cues that regulate their activation are poorly understood. Here, we establish a 3-dimensional suspension culture system containing six defined soluble factors in which primary human ductal and ductal progenitor-like cells can survive but do not proliferate. Expansion and polarization occur when cells are provided with a low concentration (5% v/v) of Matrigel, a sarcoma cell product enriched in many extracellular matrix (ECM) proteins. Screening of ECM proteins identified that collagen IV is capable of partially recapitulating the effects of Matrigel. Inhibition of integrin α1β1, a major collagen IV receptor, negates collagen IV- and Matrigel-stimulated effects. These results demonstrate that collagen IV is a key ECM protein that stimulates the expansion and polarization of human ductal and ductal progenitor-like cells via integrin α1β1 receptor signaling.

2023-06-05 | GSE233785 | GEO

Activation of adult human pancreatic ductal and ductal progenitor-like cells requires extracellular matrix protein signaling [bulkRNA-seq]

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A genome-wide analysis of human pluripotent stem cell-derived endothelial cells cultured in synthetic hydrogels compared to standard 2D or 3D cell culture platforms

Project description:The influence of 2D and 3D cell culture platforms on vascular function was investigated by comparing gene expression for human pluripotent stem cell-derived endothelial cells (H1-ECs), primary human brain vascular pericytes (pericytes), and human umbilical vein endothelial cells (HUVECs) cultured on tissue culture polystyrene (TCP, “2D”), on or in poly(ethylene glycol) (PEG) hydrogels formed via “thiol-ene” photopolymerization, and on or in gelled Matrigel. ECs cocultured with pericytes in PEG formed vascular networks with global gene expression that was highly correlated to a standard 3D Matrigel assay (Spearman’s coefficients ≥ 0.98). H1-ECs, HUVECs, and pericytes were characterized gene expression signatures associated with the cell cycle and mitosis when cultured on TCP surfaces compared to cells cultured on top of or encapsulated in PEG hydrogels or Matrigel. The proliferative signature was not necessarily a function of the 2D format, since endothelial cells cultured on PEG hydrogels were not characterized by increased proliferation or a proliferative gene signature compared to cells encapsulated in PEG hydrogels. The proliferative phenotype for H1-ECs on TCP was regulated by FAK-ERK activity, and inhibition or knockdown of ERK pathway signaling decreased proliferation and cell cycle genes while increasing expression of “3D-like” vasculature development genes. Our results suggest that cells in 2D culture adopt a highly proliferative state that interferes with normal vascular function and provides unique insight into the importance of cellular and extracellular context for in vitro tissue modeling.

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