Bas Ponsioen

Bas Ponsioen
University Medical Center Utrecht | UMC Utrecht · Department of Medical Genetics

PhD

About

50
Publications
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3,511
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Publications

Publications (50)
Article
Full-text available
CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or nonhomologous end joining (NHEJ)-mediated integration of the...
Article
Full-text available
The most successful genetically encoded calcium indicators (GECIs) employ an intensity or ratiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biological...
Article
Full-text available
We describe an optimized, cost-effective, reproducible, and robust protocol to study sprouting angiogenesis in glass-bottom 96-well plates by confocal microscopy, ideal for screening of drug or shRNA libraries. Effective and stable knockdown of gene expression in primary endothelial cells is achieved by lentiviral transduction. Dynamic behavior of...
Article
Full-text available
Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and w...
Article
Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq-a protocol that integrates live-cell imaging of tumor organoid outgrowth and w...
Preprint
Full-text available
The most successful genetically encoded calcium indicators (GECIs) employ an intensity or intensiometric readout. Despite a large calcium-dependent change in fluorescence intensity, the quantification of calcium concentrations with GECIs is problematic, which is further complicated by the sensitivity of all GECIs to changes in the pH in the biologi...
Article
Full-text available
Hematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to all blood lineages during life. HSPCs emerge from the ventral wall of the dorsal aorta (VDA) during a specific timespan in embryonic development through endothelial hematopoietic transition (EHT). We investigated the ontogeny of HSPCs in mutant zebrafish embryos la...
Article
Full-text available
Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhib...
Article
Full-text available
Accurate chromosome segregation during cell division critically depends on error correction of chromosome-spindle interactions and the spindle assembly checkpoint (SAC) [1-3]. The kinase MPS1 is an essential regulator of both processes, ensuring full chromosome biorientation before anaphase onset [3, 4]. To understand when and where MPS1 activation...
Preprint
Hematopoietic Stem and Progenitor Cells (HSPCs) are multipotent cells giving rise to all blood lineages during life. HSPCs emerge from the ventral wall of the dorsal aorta (VDA) during a specific timespan in embryonic development through endothelial hematopoietic transition (EHT). We investigated the ontogeny of HSPCs in mutant zebrafish embryos la...
Article
Enteroendocrine cells (EECs) sense intestinal content and release hormones to regulate gastrointestinal activity, systemic metabolism, and food intake. Little is known about the molecular make-up of human EEC subtypes and the regulated secretion of individual hormones. Here, we describe an organoid-based platform for functional studies of human EEC...
Conference Paper
Introduction/Background Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumour heterogeneity of OC are limited and hard to establish. Methodology We present a protocol that enables efficient derivation and long-term expansion of OC organoids....
Article
Full-text available
Ovarian cancer (OC) is a heterogeneous disease usually diagnosed at a late stage. Experimental in vitro models that faithfully capture the hallmarks and tumor heterogeneity of OC are limited and hard to establish. We present a protocol that enables efficient derivation and long-term expansion of OC organoids. Utilizing this protocol, we have establ...
Article
Full-text available
Chromosome segregation errors cause aneuploidy and genomic heterogeneity, which are hallmarks of cancer in humans. A persistent high frequency of these errors (chromosomal instability (CIN)) is predicted to profoundly impact tumor evolution and therapy response. It is unknown, however, how prevalent CIN is in human tumors. Using three-dimensional l...
Chapter
Examining cell behavior in its correct tissue context is a major challenge in cell biology. The recent development of mammalian stem cell-based organoid cultures offers exciting opportunities to visualize dynamic cellular events in a 3D tissue-like setting. We describe here an approach for live imaging of cell division processes in intestinal organ...
Article
Our understanding of the epithelial stem cell compartment of the intestine has seen tremendous progress over recent years. As a result, the crypt is progressively transforming into the primary model system to address fundamental questions about epithelial homeostasis, lineage differentiation and cancer development. Instrumental for these developmen...
Data
ImageJ/Fiji macro script: ‘Organoid movie macro’.Converts XYZT confocal data sets into analyzable 2D-movies, consisting four quadrants: depth coding, maximum projection in ‘glow’, transmitted light image and a merge between transmitted light and fluorescence. All supplementary movies were generated using this method (Figures 1 and 3 show 2 of 4 qua...
Data
Dose-response curves for patient-derived tumor organoids P18T and P18T KRASG12D as indicated.A number of biological replicates for each dose-response curve are indicated between parenthesis (first monotherapy/ second monotherapy/ combination therapy).DOI: http://dx.doi.org/10.7554/eLife.18489.016
Data
Dose-response curves for normal and normal KRASG12D organoids as indicated.Number of biological replicates for each dose-response curve are indicated between parenthesis (first monotherapy/ second monotherapy/ combination therapy).DOI: http://dx.doi.org/10.7554/eLife.18489.021
Data
Dose-response curves for panel of patient-derived tumor organoids as indicated.A number of biological replicates for each dose-response curve are indicated between parenthesis (first monotherapy/ second monotherapy/ combination therapy).DOI: http://dx.doi.org/10.7554/eLife.18489.025
Data
Dose-response curves for patient-derived tumor organoids P18T and P18T KRASG12D as indicated.A number of biological replicates for each dose-response curve are indicated between parenthesis (first monotherapy/ second monotherapy/ combination therapy).DOI: http://dx.doi.org/10.7554/eLife.18489.017
Data
ImageJ/Fiji macro script: ‘Macro Drug&release experiment’.Guides the user through XYZ stacks of organoids, acquired at various time points (days apart). Helps to find back individual organoids and, per z-slice, lets the user indicate dead H2B particles by manual drawing. All output data are summarized in excel output file. For more detail, see Mate...
Data
ImageJ/Fiji macro script: ‘Score Events macro’.Guides the user through the analysis of the event-rich organoid movies (e.g. as generated with the Organoid movie macro), by numbering and drawing indicated events (mitosis, apoptosis) in the movie and generating an overview excel file. Graphs in Figure 8D and Figure 3—figure supplement 2 were generate...
Data
ImageJ/Fiji macro script: 'Macro PI versus H2B'.Analyzes H2B-mNeon expressing organoids, that have been labelled with propidium iodide to mark dead cells/fragments. Measure for death induction is the ratio between PI-positive pixels (dead) and H2B-positive pixels (total). Thresholding values for both channels can be tweaked. Thresholded images (z-s...
Data
Dose-response data (%ATP normalized to DMSO) for all patient-derived tumor organoids as described in Figure 4—source data 1, Figure 4—source data 2, Figure 5—source data 1 and Figure 6—source data 1. DOI: http://dx.doi.org/10.7554/eLife.18489.039
Article
Gene expression-based classification systems have identified an aggressive colon cancer subtype with mesenchymal features, possibly reflecting epithelial-to-mesenchymal transition (EMT) of tumor cells. However, stromal fibroblasts contribute extensively to the mesenchymal phenotype of aggressive colon tumors, challenging the notion of tumor EMT. To...
Article
Full-text available
The small GTPase Rap1 controls the actin cytoskeleton by regulating Rho GTPase signaling. We have recently established that the Rap1 effectors Radil and Rasip1, together with the Rho GTPase activating protein ArhGAP29, mediate Rap1-induced inhibition of Rho signaling in the processes of epithelial cell spreading and endothelial barrier function. He...
Article
Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utiliz...
Article
Full-text available
In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition has to be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements or teeth. A central aspect of this concept is the tightly controlled balance betwe...
Article
Full-text available
Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter...
Article
Full-text available
Cell signaling is mostly studied in in vitro 2D-cell culture models that lack the complex in vivo environment provided by neighboring cells, soluble secreted factors and non-cellular matrix components. Given that many environmental factors control cell signaling, it comes as no surprise that in vitro observations often poorly correlate with in vivo...
Article
Full-text available
Epac1 is a guanine nucleotide exchange factor for the small G protein Rap and is involved in membrane-localized processes such as integrin-mediated cell adhesion and cell-cell junction formation. Cyclic AMP (cAMP) directly activates Epac1 by release of autoinhibition and in addition induces its translocation to the plasma membrane. Here, we show an...
Article
Full-text available
Sprouting angiogenesis requires the coordinated behaviour of endothelial cells, regulated by Notch and vascular endothelial growth factor receptor (VEGFR) signalling. Here, we use computational modelling and genetic mosaic sprouting assays in vitro and in vivo to investigate the regulation and dynamics of endothelial cells during tip cell selection...
Article
Full-text available
Chloride intracellular channel (CLIC) 4 is a soluble protein structurally related to omega-type glutathione-S-transferases (GSTs) and implicated in various biological processes, ranging from chloride channel formation to vascular tubulogenesis. However, its function(s) and regulation remain unclear. Here, we show that cytosolic CLIC4 undergoes rapi...
Article
Epac1 is a guanine nucleotide exchange factor (GEF) for the small G protein Rap and is directly activated by cyclic AMP (cAMP). Upon cAMP binding, Epac1 undergoes a conformational change that allows the interaction of its GEF domain with Rap, resulting in Rap activation and subsequent downstream effects, including integrin-mediated cell adhesion an...
Article
Full-text available
Epac1 is a guanine nucleotide exchange factor (GEF) for the small G protein Rap and is directly activated by cyclic AMP (cAMP). Upon cAMP binding, Epac1 undergoes a conformational change that allows the interaction of its GEF domain with Rap, resulting in Rap activation and subsequent downstream effects, including integrin-mediated cell adhesion an...
Article
The cover picture shows the chemical structure of an acetoxymethyl ester of 8-pCPT-2′-O-Me-cAMP (007-AM). 007-AM can pass cell membranes efficiently and is hydrolysed inside the cell by esterases to release the biologically active compound 8-pCPT-2′-O-Me-cAMP (007). Due to its low membrane permeability 007 accumulates inside the cell, where it acti...
Article
DA - 20080828 IS - 1439-7633 (Electronic) LA - ENG PT - JOURNAL ARTICLE
Article
Full-text available
We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and accept...
Article
Full-text available
Cell-cell communication through connexin43 (Cx43)-based gap junction channels is rapidly inhibited upon activation of various G protein-coupled receptors; however, the mechanism is unknown. We show that Cx43-based cell-cell communication is inhibited by depletion of phosphatidylinositol 4,5-bisphosphate (PtdIns[4,5]P(2)) from the plasma membrane. K...
Article
Gap junctions (GJ) are clusters of transmembrane channels that allow direct cell-to-cell transfer of ions and small molecules. The GJ-permeant signaling molecule cAMP is of particular interest because of its numerous cellular effects. However, to assess the biological relevance of GJ-mediated cAMP transfer, quantitative aspects must be determined....
Article
Full-text available
Glycogen synthase kinase-3 (GSK-3) is a multifunctional serine/threonine kinase that is usually inactivated by serine phosphorylation in response to extracellular cues. However, GSK-3 can also be activated by tyrosine phosphorylation, but little is known about the upstream signaling events and tyrosine kinase(s) involved. Here we describe a G prote...
Article
Full-text available
Epac1 is a guanine nucleotide exchange factor for Rap1 that is activated by direct binding of cAMP. In vitro studies suggest that cAMP relieves the interaction between the regulatory and catalytic domains of Epac. Here, we monitor Epac1 activation in vivo by using a CFP-Epac-YFP fusion construct. When expressed in mammalian cells, CFP-Epac-YFP show...
Article
Full-text available
Cells respond to extracellular cues via receptor signaling. In this manner, cellular behavior is under strict control of hormones, growth factors or neurotransmitters. Binding of a ligand to its cognate receptor triggers a cascade of intracellular signaling events leading to, for instance, activation of transcription factors, regulation of metaboli...

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