Publications

Tissue stem cell (TSC)-derived epithelial organoids are typically cultured in Matrigel [T. Sato et al. , Nature 459 , 262–265 (2009)], an extracellular matrix-like hydrogel produced from Engelbreth–Holm–Swarm sarcoma cells. This tumor is grown in the mouse abdomen [R. W. Orkin et al. , J. Exp. Med. 145 , 204–220 (1977)]. Previously, we demonstrated that the Yersinia membrane protein Invasin, coated on transwells, replaces Matrigel by activating β1-integrins, allowing long-term expansion of primary epithelial cells as 2D organoid sheets [J. J. A. P. M. Wijnakker et al. , Proc. Natl. Acad. Sci. U.S.A. 122 , e2420595121 (2025)]. Here, we functionalize a synthetic polyisocyanide (PIC) hydrogel with the integrin-activating domain of Invasin (INV). PIC hydrogels are soluble at 4 °C and form a gel at 37 °C [P. H. J. Kouwer et al. , Nature 493 , 651–655 (2013)]. When INV is covalently linked to PIC, the resulting hydrogel supports multipassage 3D growth of human intestinal and airway organoids. Self-renewal, polarization, and differentiation are maintained. The 3D swelling assay for cystic fibrosis drug testing (S. F. Boj et al. , J. Vis. Exp. (2017), 10.3791/55159] was validated using PIC-INV. With PIC-INV hydrogels, we establish a fully defined and animal-free system for 3D TSC-derived organoid culture.

The purpose of this 3-wave study is to examine the micro process of how employees adapt to change over time. We combined Conservation of Resources theory with insights from the organizational change literature to study employees in a Dutch police district undergoing reorganization. A model was tested where employee adaptability, operationalized by the presence of resources, predicts individual adaptive attitudes as well as adaptive behavior over time. Change information was included as a contextual change resource and meaning-making as a personal change resource. The research design allowed for examining longitudinal relationships by capturing data (1) before (Time 1), (2) during (Time 2), and (3) after change implementation (Time 3). We expected adaptability before and during change implementation to predict adaptive attitudes and adaptive behavior (both during and after change implementation). In addition, different indirect effects were tested. Structural equation modeling analyses supported most of the hypothesized relationships between resources and outcomes, although relationships differed from T1 to T2 and from T2 to T3. T1 change information triggered T2 meaning-making which translated into T3 adaptive behavior. The relevance of meaning-making and change information as important predictors of adaptive behavior is emphasized. Implications of these longitudinal findings are discussed.

ABSTRACT SARS-CoV-2 can enter cells after its spike protein is cleaved by either type II transmembrane serine proteases (TTSPs), like TMPRSS2, or cathepsins. It is now widely accepted that the Omicron variant uses TMPRSS2 less efficiently and instead enters cells via cathepsins, but these findings have yet to be verified in more relevant cell models. Although we could confirm efficient cathepsin-mediated entry for Omicron in a monkey kidney cell line, experiments with protease inhibitors showed that Omicron (BA.1 and XBB1.5) did not use cathepsins for entry into human airway organoids and instead utilized TTSPs. Likewise, CRISPR-edited intestinal organoids showed that entry of Omicron BA.1 relied on the expression of the serine protease TMPRSS2 but not cathepsin L or B. Together, these data force us to rethink the concept that Omicron has adapted to cathepsin-mediated entry and indicate that TTSP inhibitors should not be dismissed as prophylactic or therapeutic antiviral strategy against SARS-CoV-2. IMPORTANCE Coronavirus entry relies on host proteases that activate the viral fusion protein, spike. These proteases determine the viral entry route, tropism, host range, and can be attractive drug targets. Whereas earlier studies using cell lines suggested that the Omicron variant of SARS-CoV-2 has changed its protease usage, from cell surface type II transmembrane serine proteases (TTSPs) to endosomal cathepsins, we report that this is not the case in human airway and intestinal organoid models, suggesting that host TTSP inhibition is still a viable prophylactic or therapeutic antiviral strategy against current SARS-CoV-2 variants and highlighting the importance of relevant human in vitro cell models.

<p>Differential sgRNA enrichment from CRISPR-Cas9 genome-wide screens</p>

Abstract Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids (IOs) are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses.