Isolation and propagation of primary human cholangiocyte organoids for the generation of bioengineered biliary tissue.

Pediatric liver transplantation is often required as a consequence of biliary disorders because of the lack of alternative treatments for repairing or replacing damaged bile ducts. To address the lack of availability of pediatric livers suitable for transplantation, we developed a protocol for generating bioengineered biliary tissue suitable for biliary reconstruction. Our platform allows the derivation of cholangiocyte organoids (COs) expressing key biliary markers and retaining functions of primary extra- or intrahepatic duct cholangiocytes within 2 weeks of isolation. COs are subsequently seeded on polyglycolic acid (PGA) scaffolds or densified collagen constructs for 4 weeks to generate bioengineered tissue retaining biliary characteristics. Expertise in organoid culture and tissue engineering is desirable for optimal results. COs correspond to mature functional cholangiocytes, differentiating our method from alternative organoid systems currently available that propagate adult stem cells. Consequently, COs provide a unique platform for studies in biliary physiology and pathophysiology, and the resulting bioengineered tissue has broad applications for regenerative medicine and cholangiopathies

Cardiovascular ACE2 receptor expression in patients undergoing heart transplantation

Abstract: Aims: Membrane‐bound angiotensin‐converting enzyme (ACE)2 is the main cellular access point for SARS‐CoV‐2, but its expression and the effect of ACE inhibition have not been assessed quantitatively in patients with heart failure. The aim of this study was to characterize membrane‐bound ACE2 expression in the myocardium and myocardial vasculature in patients undergoing heart transplantation and to assess the effect of pharmacological ACE inhibition. Methods and results: Left ventricular (LV) tissue was obtained from 36 explanted human hearts from patients undergoing heart transplantation. Immunohistochemical staining with antibodies directed against ACE2 co‐registered with cardiac troponin T (cTnT) and α‐smooth muscle cell actin (SMA) was performed across the entire cohort. ACE2 receptor expression was quantitatively assessed throughout the myocardium and vasculature. ACE2 was consistently expressed throughout the LV myocardium (28.3% ± 22.2% of cardiomyocytes). ACE2 expression was also detected in small calibre blood vessels (range, 2–9 μm), albeit at quantitatively much lower levels (5% ± 9% of blood vessels). There was no significant difference in ACE2 expression between patients receiving ACE inhibitors prior to transplantation and ACE inhibitor‐negative controls (P > 0.05). ACE2 expression did not differ significantly between the different diagnostic groups as the underlying reason for heart transplantation (ANOVA > 0.05). N‐terminal pro‐brain natriuretic peptide (NT‐proBNP) (R2 = 0.37, P = 0.0006) and pulmonary capillary wedge pressure (PCWP) (R2 = 0.13, P = 0.043) assessed by right heart catheterization were significantly correlated with greater ACE2 expression in cardiomyocytes. Conclusions: These data provide a comprehensive characterization of membrane‐bound cardiac ACE2 expression in patients with heart failure with no demonstrable effect exerted by ACE inhibitors

Cholangiocyte organoids can repair bile ducts after transplantation in the human liver.

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

Developing novel prophylactic approaches against SARS-CoV-2 infection

Prevention of SARS-CoV-2 entry into cells through the modulation of viral host receptors, such as ACE2, could represent a new chemoprophylactic and therapeutic approach for COVID- 19 complementing vaccination. However, the mechanisms controlling ACE2 expression remain elusive, partly due to low ACE2 levels in experimental models. To address this challenge, I use biliary epithelial cell (cholangiocyte), one of the cell types with the highest ACE2 levels, to dissect the molecular mechanisms controlling ACE2 expression. I identify the farnesoid X receptor (FXR) as a direct regulator of ACE2 transcription in multiple COVID19- affected tissues, including the gastrointestinal and respiratory systems. I use this knowledge to optimise current experimental models for SARS-CoV-2 infection and I demonstrate that reduction of FXR signalling, with the over-the-counter compound z-guggulsterone (ZGG) and the off-patent drug ursodeoxycholic acid (UDCA), downregulates ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. I also show that UDCA-mediated ACE2 downregulation reduces susceptibility to SARS-CoV- 2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. I then illustrate that UDCA reduces ACE2 expression in the nasal epithelium in humans. Finally, I present a retrospective analysis using an independent patient cohort which identify a correlation between UDCA treatment and positive clinical outcomes following SARS-CoV-2 infection, including hospitalisation, ICU admission and death using retrospective COVID-19 registry data, and confirm these findings in a second independent cohort of liver transplant recipients. In conclusion, with the work described in this dissertation I identify a novel function of FXR in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, thereby paving the road for future clinical trials

Organoids and regenerative hepatology.

The burden of liver diseases is increasing worldwide, with liver transplantation remaining the only treatment option for end-stage liver disease. Regenerative medicine holds great potential as a therapeutic alternative, aiming to repair or replace damaged liver tissue with healthy functional cells. The properties of the cells used are critical for the efficacy of this approach. The advent of liver organoids has not only offered new insights into human physiology and pathophysiology, but also provided an optimal source of cells for regenerative medicine and translational applications. Here, we discuss various historical aspects of 3D organoid culture, how it has been applied to the hepatobiliary system, and how organoid technology intersects with the emerging global field of liver regenerative medicine. We outline the hepatocyte, cholangiocyte, and nonparenchymal organoids systems available and discuss their advantages and limitations for regenerative medicine as well as future directions

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts tropism and fusogenicity

SARS-CoV-2オミクロン株による中和抗体回避と感染指向性の変化. 京都大学プレスリリース. 2022-02-03.The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and bears multiple spike mutations2. Here we show that Omicron spike has higher affinity for ACE2 compared to Delta as well as a marked change of antigenicity conferring significant evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralising antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralisation. Importantly, antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lower airway organoids, lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared to Delta. Replication differences mapped to entry efficiency using spike pseudotyped virus (PV) assays. The defect for Omicron PV to enter specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and knock down of TMPRSS2 impacted Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently utilises the cellular protease TMPRSS2 that promotes cell entry via plasma membrane fusion, with greater dependency on cell entry via the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to utilise TMPRSS2, syncytium formation by the Omicron spike was markedly impaired compared to the Delta spike. Omicron’s less efficient spike cleavage at S1/S2 is associated with shift in cellular tropism away from TMPRSS2 expressing cells, with implications for altered pathogenesis

Altered TMPRSS2 usage by SARS-CoV-2 Omicron impacts infectivity and fusogenicity.

The SARS-CoV-2 Omicron BA.1 variant emerged in 20211 and has multiple mutations in its spike protein2. Here we show that the spike protein of Omicron has a higher affinity for ACE2 compared with Delta, and a marked change in its antigenicity increases Omicron's evasion of therapeutic monoclonal and vaccine-elicited polyclonal neutralizing antibodies after two doses. mRNA vaccination as a third vaccine dose rescues and broadens neutralization. Importantly, the antiviral drugs remdesivir and molnupiravir retain efficacy against Omicron BA.1. Replication was similar for Omicron and Delta virus isolates in human nasal epithelial cultures. However, in lung cells and gut cells, Omicron demonstrated lower replication. Omicron spike protein was less efficiently cleaved compared with Delta. The differences in replication were mapped to the entry efficiency of the virus on the basis of spike-pseudotyped virus assays. The defect in entry of Omicron pseudotyped virus to specific cell types effectively correlated with higher cellular RNA expression of TMPRSS2, and deletion of TMPRSS2 affected Delta entry to a greater extent than Omicron. Furthermore, drug inhibitors targeting specific entry pathways3 demonstrated that the Omicron spike inefficiently uses the cellular protease TMPRSS2, which promotes cell entry through plasma membrane fusion, with greater dependency on cell entry through the endocytic pathway. Consistent with suboptimal S1/S2 cleavage and inability to use TMPRSS2, syncytium formation by the Omicron spike was substantially impaired compared with the Delta spike. The less efficient spike cleavage of Omicron at S1/S2 is associated with a shift in cellular tropism away from TMPRSS2-expressing cells, with implications for altered pathogenesis

FXR inhibition may protect from SARS-CoV-2 infection by reducing ACE2.

Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. 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