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As one of the primary points of entry of xenobiotic substances and infectious agents into the body, the lungs are subject to a range of dysfunctions and diseases that together account for a significant number of patient deaths. In view of this, there is an outstanding need for in vitro systems in which to assess the impact of both infectious agents and xenobiotic substances of the lungs. To address this issue, we have developed a protocol to generate airway epithelial basal‐like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways. Basal‐like cells generated in this study were cultured on transwell inserts to allow formation of a confluent monolayer and then exposed to an air‐liquid interface to induce differentiation into a pseudostratified epithelial construct with a marked similarity to the upper airway epithelium in vivo. These constructs contain the component cell types required of an epithelial model system, produce mucus and functional cilia, and can support SARS‐CoV‐2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways. This method offers a readily accessible and highly scalable protocol for the manufacture of upper airway models that could find applications in development of therapies for respiratory viral infections and the assessment of drug toxicity on the human lungs., We have developed a protocol to generate airway epithelial basal‐like cells from induced pluripotent stem cells, which simplifies the manufacture of cellular models of the human upper airways that are capable of supporting SARS‐CoV‐2 infection/replication and the secretion of cytokines in a manner similar to that of in vivo airways.

Fibrosis is a common pathological feature of chronic disease. Deletion of the NF-κB subunit c-Rel limits fibrosis in multiple organs, although the mechanistic nature of this protection is unresolved. Using cell-specific gene-targeting manipulations in mice undergoing liver damage, we elucidate a critical role for c-Rel in controlling metabolic changes required for inflammatory and fibrogenic activities of hepatocytes and macrophages and identify Pfkfb3 as the key downstream metabolic mediator of this response. Independent deletions of Rel in hepatocytes or macrophages suppressed liver fibrosis induced by carbon tetrachloride, while combined deletion had an additive anti-fibrogenic effect. In transforming growth factor-β1-induced hepatocytes, c-Rel regulates expression of a pro-fibrogenic secretome comprising inflammatory molecules and connective tissue growth factor, the latter promoting collagen secretion from HMs. Macrophages lacking c-Rel fail to polarize to M1 or M2 states, explaining reduced fibrosis in RelΔLysM mice. Pharmacological inhibition of c-Rel attenuated multi-organ fibrosis in both murine and human fibrosis. In conclusion, activation of c-Rel/Pfkfb3 in damaged tissue instigates a paracrine signalling network among epithelial, myeloid and mesenchymal cells to stimulate fibrogenesis. Targeting the c-Rel-Pfkfb3 axis has potential for therapeutic applications in fibrotic disease. ispartof: NATURE METABOLISM vol:2 issue:11 ispartof: location:Germany status: published

Correction to: Nature Metabolism https://doi.org/10.1038/s42255-020-00306-2, published online 9 November 2020. In the version of this article initially published, in the ×40 diseased human kidney images in Supplementary Fig. 1, the FSGS image duplicated the DN image. The error has been corrected in the HTML version of the article.

Mutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31 /- mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.

SummaryMutations in pre-mRNA processing factors (PRPFs) cause 40% of autosomal dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed PRPFs cause retinal disease. To understand the molecular basis of this phenotype, we have generated RP type 11 (PRPF31-mutated) patient-specific retinal organoids and retinal pigment epithelium (RPE) from induced pluripotent stem cells (iPSC). Impaired alternative splicing of genes encoding pre-mRNA splicing proteins occurred in patient-specific retinal cells and Prpf31+/− mouse retinae, but not fibroblasts and iPSCs, providing mechanistic insights into retinal-specific phenotypes of PRPFs. RPE was the most affected, characterised by loss of apical-basal polarity, reduced trans-epithelial resistance, phagocytic capacity, microvilli, and cilia length and incidence. Disrupted cilia morphology was observed in patient-derived-photoreceptors that displayed progressive features associated with degeneration and cell stress. In situ gene-editing of a pathogenic mutation rescued key structural and functional phenotypes in RPE and photoreceptors, providing proof-of-concept for future therapeutic strategies.eTOCPRPF31 is a ubiquitously expressed pre-mRNA processing factor that when mutated causes autosomal dominant RP. Using a patient-specific iPSC approach, Buskin and Zhu et al. show that retinal-specific defects result from altered splicing of genes involved in the splicing process itself, leading to impaired splicing, loss of RPE polarity and diminished phagocytic ability as well as reduced cilia incidence and length in both photoreceptors and RPE.HighlightsSuccessful generation of iPSC-derived RPE and photoreceptors from four RP type 11 patientsRPE cells express the mutant PRPF31 protein and show the lowest expression of wildtype proteinPRPF31 mutations result in altered splicing of genes involved in pre-mRNA splicing in RPE and retinal organoidsPrpf31 haploinsufficiency results in altered splicing of genes involved in pre-mRNA splicing in mouse retinaRPE cells display loss of polarity, reduced barrier function and phagocytosisPhotoreceptors display shorter and fewer cilia and degenerative featuresRPE cells display most abnormalities suggesting they might be the primary site of pathogenesisIn situ gene editing corrects the mutation and rescues key phenotypes

Acquiring a mechanistic understanding of the processes underlying the renal clearance of drug molecules in man has been hampered by a lack of robust in vitro models of human proximal tubules. Several human renal epithelial cell lines derived from the renal cortex are available, but few have been characterised in detail in terms of transporter expression. This includes the HK-2 proximal tubule cell line, which has been used extensively as a model of nephrotoxicity. The aim of this study was to investigate the expression and function of drug transporters in HK-2 cells and their suitability as an in vitro model of the human proximal tubule. qPCR showed no mRNA expression of the SLC22 transporter family (OAT1, OAT3, OCT2) in HK-2 cells compared to renal cortex samples. In contrast, SLC16A1 (MCT1), which is important in the uptake of monocarboxylates, and SLCO4C1 (OATP4C1) were expressed in HK-2 cells. The functional expression of these transporters was confirmed by uptake studies using radiolabelled prototypic substrates DL-lactate and digoxin, respectively. The mRNA expression of apical membrane efflux transporters ABCB1 (MDR1) and several members of the ABCC family (multidrug resistance proteins, MRPs) was shown by qPCR. ABCG1 (BCRP) was not detected. The efflux of Hoechst 33342, a substrate for MDR1, was blocked by MDR1 inhibitor cyclosporin A, suggesting the functional expression of this transporter. Similarly, the efflux of the MRP-specific fluorescent dye glutathione methylfluorescein was inhibited by the MRP inhibitor MK571. Taken together, the results of this study suggest that HK-2 cells are of limited value as an in vitro model of drug transporter expression in the human proximal tubule.