Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. We address this challenge using cholangiocyte organoids in the context of cholangiopathies, ...which represent a key reason for liver transplantation. Using single-cell RNA sequencing, we show that primary human cholangiocytes display transcriptional diversity that is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof of principle that cholangiocyte organoids can be used to repair human biliary epithelium.
Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)
, could represent a new chemoprophylactic approach for COVID-19 that complements ...vaccination
. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.
The signalling pathways that maintain primed human pluripotent stem cells (hPSCs) have been well characterised, revealing a critical role for TGFβ/Activin/Nodal signalling. In contrast, the ...signalling requirements of naive human pluripotency have not been fully established. Here, we demonstrate that TGFβ signalling is required to maintain naive hPSCs. The downstream effector proteins - SMAD2/3 - bind common sites in naive and primed hPSCs, including shared pluripotency genes. In naive hPSCs, SMAD2/3 additionally bind to active regulatory regions near to naive pluripotency genes. Inhibiting TGFβ signalling in naive hPSCs causes the downregulation of SMAD2/3-target genes and pluripotency exit. Single-cell analyses reveal that naive and primed hPSCs follow different transcriptional trajectories after inhibition of TGFβ signalling. Primed hPSCs differentiate into neuroectoderm cells, whereas naive hPSCs transition into trophectoderm. These results establish that there is a continuum for TGFβ pathway function in human pluripotency spanning a developmental window from naive to primed states.
ABSTRACT
The functional integrity of the intestinal epithelial barrier relies on tight coordination of cell proliferation and migration, with failure to regulate these processes resulting in disease. ...It is not known whether cell proliferation is sufficient to drive epithelial cell migration during homoeostatic turnover of the epithelium. Nor is it known precisely how villus cell migration is affected when proliferation is perturbed. Some reports suggest that proliferation and migration may not be related while other studies support a direct relationship. We used established cell‐tracking methods based on thymine analog cell labeling and developed tailored mathematical models to quantify cell proliferation and migration under normal conditions and when proliferation is reduced and when it is temporarily halted. We found that epithelial cell migration velocities along the villi are coupled to cell proliferation rates within the crypts in all conditions. Furthermore, halting and resuming proliferation results in the synchronized response of cell migration on the villi. We conclude that cell proliferation within the crypt is the primary force that drives cell migration along the villus. This methodology can be applied to interrogate intestinal epithelial dynamics and characterize situations in which processes involved in cell turnover become uncoupled, including pharmacological treatments and disease models.—Parker, A., Maclaren, O. J., Fletcher, A. G., Muraro, D., Kreuzaler, P. A., Byrne, H. M., Maini, P. K., Watson, A. J. M., Pin, C. Cell proliferation within small intestinal crypts is the principal driving force for cell migration on villi. FASEB J. 31, 636–649 (2017). www.fasebj.org
The intestinal epithelial monolayer, at the boundary between microbes and the host immune system, plays an important role in the development of inflammatory bowel disease (IBD), particularly as a ...target and producer of pro-inflammatory TNF. Chronic overexpression of TNF leads to IBD-like pathology over time, but the mechanisms driving early pathogenesis events are not clear. We studied the epithelial response to inflammation by combining mathematical models with in vivo experimental models resembling acute and chronic TNF-mediated injury. We found significant villus atrophy with increased epithelial cell death along the crypt-villus axis, most dramatically at the villus tips, in both acute and chronic inflammation. In the acute model, we observed overexpression of TNF receptor I in the villus tip rapidly after TNF injection and concurrent with elevated levels of intracellular TNF and rapid shedding at the tip. In the chronic model, sustained villus atrophy was accompanied by a reduction in absolute epithelial cell turnover. Mathematical modelling demonstrated that increased cell apoptosis on the villus body explains the reduction in epithelial cell turnover along the crypt-villus axis observed in chronic inflammation. Cell destruction in the villus was not accompanied by changes in proliferative cell number or division rate within the crypt. Epithelial morphology and immunological changes in the chronic setting suggest a repair response to cell damage although the villus length is not recovered. A better understanding of how this state is further destabilised and results in clinical pathology resembling IBD will help identify suitable pathways for therapeutic intervention.
As multicellular organisms grow, positional information is continually needed to regulate the pattern in which cells are arranged. In the Arabidopsis root, most cell types are organized in a radially ...symmetric pattern; however, a symmetry-breaking event generates bisymmetric auxin and cytokinin signaling domains in the stele. Bidirectional cross-talk between the stele and the surrounding tissues involving a mobile transcription factor, SHORT ROOT (SHR), and mobile microRNA species also determines vascular pattern, but it is currently unclear how these signals integrate. We use a multicellular model to determine a minimal set of components necessary for maintaining a stable vascular pattern. Simulations perturbing the signaling network show that, in addition to the mutually inhibitory interaction between auxin and cytokinin, signaling through SHR, microRNA 165/6, and PHABULOSA is required to maintain a stable bisymmetric pattern. We have verified this prediction by observing loss of bisymmetry in shr mutants. The model reveals the importance of several features of the network, namely the mutual degradation of microRNA165/6 and PHABULOSA and the existence of an additional negative regulator of cytokinin signaling. These components form a plausible mechanism capable of patterning vascular tissues in the absence of positional inputs provided by the transport of hormones from the shoot.
NOD2 and TLR2 recognize components of bacterial cell wall peptidoglycan and direct defense against enteric pathogens. CD8
T cells are important for immunity to such pathogens but how NOD2 and TLR2 ...induce antigen specific CD8
T cell responses is unknown. Here, we define how these pattern recognition receptors (PRRs) signal in primary dendritic cells (DCs) to influence MHC class I antigen presentation. We show NOD2 and TLR2 phosphorylate PI31 via TBK1 following activation in DCs. PI31 interacts with TBK1 and Sec16A at endoplasmic reticulum exit sites (ERES), which positively regulates MHC class I peptide loading and immunoproteasome stability. Following NOD2 and TLR2 stimulation, depletion of PI31 or inhibition of TBK1 activity
impairs DC cross-presentation and CD8
T cell activation. DCs from Crohn's patients expressing
polymorphisms show dysregulated cross-presentation and CD8
T cell responses. Our findings reveal unidentified mechanisms that underlie CD8
T cell responses to bacteria in health and in Crohn's.
Recent Genome-Wide Association Studies (GWAS) have revealed numerous Crohn's disease susceptibility genes and a key challenge now is in understanding how risk polymorphisms in associated genes might ...contribute to development of this disease. For a gene to contribute to disease phenotype, its risk variant will likely adversely communicate with a variety of other gene products to result in dysregulation of common signaling pathways. A vital challenge is to elucidate pathways of potentially greatest influence on pathological behaviour, in a manner recognizing how multiple relevant genes may yield integrative effect. In this work we apply mathematical analysis of networks involving the list of recently described Crohn's susceptibility genes, to prioritise pathways in relation to their potential development of this disease. Prioritisation was performed by applying a text mining and a diffusion based method (GRAIL, GPEC). Prospective biological significance of the resulting prioritised list of proteins is highlighted by changes in their gene expression levels in Crohn's patients intestinal tissue in comparison with healthy donors.
Cell cycle progression and cell fate decisions are closely linked in human pluripotent stem cells (hPSCs). However, the study of these interplays at the molecular level remains challenging due to the ...lack of efficient methods allowing cell cycle synchronization of large quantities of cells. Here, we screened inhibitors of cell cycle progression and identified nocodazole as the most efficient small molecule to synchronize hPSCs in the G2/M phase. Following nocodazole treatment, hPSCs remain pluripotent, retain a normal karyotype and can successfully differentiate into the three germ layers and functional cell types. Moreover, genome-wide transcriptomic analyses on single cells synchronized for their cell cycle and differentiated toward the endoderm lineage validated our findings and showed that nocodazole treatment has no effect on gene expression during the differentiation process. Thus, our synchronization method provides a robust approach to study cell cycle mechanisms in hPSCs.
Display omitted
•Nocodazole can enrich cells in the G2/M, G1, and S phases of the cell cycle•Treatment with nocodazole does not affect pluripotency maintenance•hPSCs can efficiently form functional cell types after nocodazole treatment•Nocodazole treatment allows genome-wide analyses of synchronous populations
In this study, Vallier and colleagues show that small molecule cell cycle inhibitors can be used to successfully synchronize and enrich hPSCs in different cell cycle phases. Particularly, the G2/M inhibitor nocodazole successfully enriched cells in G2/M, G1, and S phases of the cell cycle, without affecting pluripotency and differentiation capacity as shown by molecular and genome-wide single-cell RNA-seq analyses.
Inflammatory bowel disease (IBD) consists of two main disease-subtypes, Crohn's disease (CD) and ulcerative colitis (UC); these subtypes share overlapping genetic and clinical features. Genome-wide ...microarray data enable unbiased documentation of alterations in gene expression that may be disease-specific. As genetic diseases are believed to be caused by genetic alterations affecting the function of signalling pathways, module-centric optimisation algorithms, whose aim is to identify sub-networks that are dys-regulated in disease, are emerging as promising approaches.
In order to account for the topological structure of molecular interaction networks, we developed an optimisation algorithm that integrates databases of known molecular interactions with gene expression data; such integration enables identification of differentially regulated network modules. We verified the performance of our algorithm by testing it on simulated networks; we then applied the same method to study experimental data derived from microarray analysis of CD and UC biopsies and human interactome databases. This analysis allowed the extraction of dys-regulated subnetworks under different experimental conditions (inflamed and uninflamed tissues in CD and UC). Optimisation was performed to highlight differentially expressed network modules that may be common or specific to the disease subtype.
We show that the selected subnetworks include genes and pathways of known relevance for IBD; in particular, the solutions found highlight cross-talk among enriched pathways, mainly the JAK/STAT signalling pathway and the EGF receptor signalling pathway. In addition, integration of gene expression with molecular interaction data highlights nodes that, although not being differentially expressed, interact with differentially expressed nodes and are part of pathways that are relevant to IBD. The method proposed here may help identifying dys-regulated sub-networks that are common in different diseases and sub-networks whose dys-regulation is specific to a particular disease.
PDF
