Publications

Isogenic disease models, such as genetically engineered organoids, provide insight into the impact of genetic variants on organ function. Here, we present a protocol to create isogenic disease models from adult stem cell-derived organoids using next-generation CRISPR tools. We describe steps for single guide RNA (sgRNA) design and cloning, electroporation, and selecting electroporated cells. We then detail procedures for clonal line generation. Next-generation CRISPR tools do not require double-stranded break (DSB) induction for their function, thus simplifying in vitro disease model generation. For complete details on the use and execution of this protocol, please refer to Geurts et al.¹^,².

In the past five years, pluripotent stem cell (PSC)-derived kidney organoids and adult stem or progenitor cell (ASC)-based kidney tubuloids have emerged as advanced in vitro models of kidney development, physiology, and disease. PSC-derived organoids mimic nephrogenesis. After differentiation towards the kidney precursor tissues ureteric bud and metanephric mesenchyme, their reciprocal interaction causes self-organization and patterning in vitro to generate nephron structures that resemble the fetal kidney. ASC tubuloids on the other hand recapitulate renewal and repair in the adult kidney tubule and give rise to long-term expandable and genetically stable cultures that consist of adult proximal tubule, loop of Henle, distal tubule, and collecting duct epithelium. Both organoid types hold great potential for: (1) studies of kidney physiology, (2) disease modeling, (3) high-throughput screening for drug efficacy and toxicity, and (4) regenerative medicine. Currently, organoids and tubuloids are successfully used to model hereditary, infectious, toxic, metabolic, and malignant kidney diseases and to screen for effective therapies. Furthermore, a tumor tubuloid biobank was established, which allows studies of pathogenic mutations and novel drug targets in a large group of patients. In this review, we discuss the nature of kidney organoids and tubuloids and their current and future applications in science and medicine.

Supplementary Methods from Cyclin D2–Cyclin-Dependent Kinase 4/6 Is Required for Efficient Proliferation and Tumorigenesis following Apc Loss

The aim of the present study was to investigate relationships between Effort-Reward Imbalance (ERI) and employee adverse health (i.e., psychosomatic complaints, physical symptoms, and exhaustion). The Effort-Reward Imbalance Model was extended by discriminating three specific rewards separately (i.e., salary, esteem, and job security). A sample of 167 health-care workers was used to test the relationship between ERI (i.e., high effort accompanied with low reward) and employee adverse health with multiple univariate logistic regression analyses. Separate analyses were carried out with a composite reward scale and with each of the three specific reward scales as independent variables. The results indicate that the health outcomes of ERI vary depending on the specific reward that was used. Generally, the most adverse health effects were found for employees who reported both high efforts and low rewards, thus supporting the ERI Model. Moreover, the strongest effects of ERI on employee health were found when esteem was used as a reward indicator. Job security also appeared to be an important reward in this respect, whereas salary did not have strong effects as a result of ERI. Although the ERI Model was merely supported by the results, current findings also indicate it is important to separate different kinds of rewards, especially in health-care work in order to capture the complexity of working with clients as well.

Intestinal epithelial cells (IECs) are capable of mounting an adequate antimicrobial inflammatory response to pathogens while tolerating commensals. The underlying regulatory mechanisms of immune sensitivity remain incompletely understood, particularly in the context of human IECs. To enhance our understanding of the immune response of IECs to bacterial epithelial barrier breach, we investigated whether epithelial responsiveness is contingent on cell identity and cell polarization. We exposed human intestinal organoids to bacterial antigens to study their immune responses. Notable discrepancies were observed in the specific reactions exhibited by intestinal stem cells (ISCs) and enterocytes. It was determined that basolateral exposure of IECs to bacterial antigens resulted in a robust response, whereas apical exposure elicited a significantly more modest response. We identified ISCs as the responders, while the reaction of enterocytes was found to be attenuated. The regulation of bacterial responsiveness in enterocytes occurs at multiple levels, including the modulation of NFκB activation and post-transcriptional control of mRNA stability. Our findings demonstrate that differentiated non-responsive enterocytes can be sensitized to bacterial antigens through the activation of the WNT pathway. These findings extend the crucial role of WNT signaling for intestinal epithelial homeostasis and regulation of stem cell maintenance, proliferation, differentiation, and tissue architecture in the gut. Additionally, they reveal a new function of WNT signaling in regulating microbial responses within the intestinal environment.

Abstract The lack of a robust system to reproducibly propagate HRV-C, a family of viruses refractory to cultivation in standard cell lines, has substantially hindered our understanding of this common respiratory pathogen. We sought to develop an organoid-based system to reproducibly propagate HRV-C, and characterize virus-host interaction using respiratory organoids. We demonstrate that airway organoids sustain serial virus passage with the aid of CYT387-mediated immunosuppression, whereas nasal organoids that more closely simulate the upper airway achieve this without any intervention. Nasal organoids are more susceptible to HRV-C than airway organoids. Intriguingly, upon HRV-C infection, we observe an innate immune response that is stronger in airway organoids than in nasal organoids, which is reproduced in a Poly(I:C) stimulation assay. Treatment with α-CDHR3 and antivirals significantly reduces HRV-C viral growth in airway and nasal organoids. Additionally, an organoid-based immunofluorescence assay is established to titrate HRV-C infectious particles. Collectively, we develop an organoid-based system to reproducibly propagate the poorly cultivable HRV-C, followed by a comprehensive characterization of HRV-C infection and innate immunity in physiologically active respiratory organoids. The organoid-based HRV-C infection model can be extended for developing antiviral strategies. More importantly, our study has opened an avenue for propagating and studying other uncultivable human and animal viruses.

Additional file 17. Table S16