Periodic Reporting for period 5 - New Chol (Development of New therapies against cholangiopathies.)
Reporting period: 2022-07-01 to 2023-11-30
Cholangiopathies regroup diseases affecting cholangiocytes which are the main cell type of the biliary tract. These disorders range from inherited (Cystic Fibrosis) and developmental (Alagille Syndrome, Biliary Atresia) to autoimmune (Primary Biliary Cirrhosis), idiopathic (Primary Sclerosing Cholangitis) and drug or toxin induced diseases. Cholangiopathies result in toxic bile accumulation in the liver inducing cell death and ultimately cirrhosis. They carry high morbidity and mortality, accounting for up to a third of chronic liver disorders. Whole liver transplantation remains the main treatment. However, organ transplant requires immunosuppression with significant side effects and an increasing number of patients die while on the transplant list due to the shortage of suitable donors. The absence of physiologically relevant in vitro systems to model and to study cholangiopathies have prevented the development of new therapeutics. Similarly, the difficulty to grow biliary cells in vitro has so far blocked the advance of cell-based therapies against diseases affecting cholangiocytes. During the NewChol program, we have systematically addressed these challenges limiting the new therapies against cholangiopathies by developing a novel and innovative program of translational research focusing on cholangiocytes.
The overall objective of the New-Chol project was to address these challenges. We first investigated the cellular diversity of cholangiocytes lining the biliary tree especially between extra- and intra-hepatic ducts. For that, we collected single cell from organ donors from 3 regions of the biliary tree (extra-hepatic (Gallbladder and common bile duct) and Intra-hepatic). We then performed bulk RNA-Seq [Rimland et al., Hepatology 2020 ] and Single Cell RNA-Seq [Samapaziotis et al., Science 2021] analyses to map the cellular diversity of the biliary epithelium. These experiments shows that cholangiocytes express specific markers in function of their location in the tree. Importantly, cholangiocytes maintain a shared transcriptome trough the tree while most regional markers were expressed as gradient along the ducts. Overall, these data suggest that the microenvironment and niche factors could impose a pattern of expression in cholangiocytes.
While we collected primary cells for transcriptomic analyses, we also derived Cholangiocytes organoids from the same samples. The cells collected were then grown in 3D conditions using culture media specially developed for this program [Samapaziotis et al., Nat. Medicine 2017]. The resulting organoids were characterised as described [Tysoe et al., Nat. Protocols 2019] and their transcriptome compared to those of primary tissue [Sampaziotis et al., Nat Protocols 2017]. These experiments show that primary cholangiocyte organoids maintain their functional activities for bile acid and drug transports. However, organoids derived from the different region of the biliary tree lose their regionalisation [Sampaziotis et al., Science 2021]. Regionalisation could be re-established in organoids by adding in our culture conditions niche factors such as bile acids. Thus, our results have established that cholangiocytes can adopt different regional state in function of their microenvironment. This plasticity could explain in part the regionality of cholangiopathies. These results were also used to define the nomenclature of cholangiocytes organoids in the hepatology field [Marsee et al., Cell Stem Cell 2021].
We then explored the interest of cholangiocyte organoids to model cholangiopathies in vitro. Fort that, we showed that monogenetic disorders such as Alagille Syndrome can be modelled in vitro by blocking Notch signalling [Sampaziotis et al., Nat. Biotech 2015] or by genome editing using protocol developed for hiPSCs [Bertero et al., Development 2016]. We also validate the interest of intrahepatic cholangiocytes organoids (ICOs) to study idiopathic cholangiopathies. Finally, we have established a platform to study ductular reaction (DR) a process which is commonly observed during the progression of chronic liver diseases such as Metabolic dysfunction-associated fatty liver disease (MASLD). DR involves the proliferation of cholangiocytes and a profound remodelling of the biliary tree resulting in further disease progression. However, it has been suggested that DR could also represent a regenerative process aiming to repair the liver. In sum, we have shown that ICOs grown in vitro can be used to model DR and the associated regenerative processes [Gribben et al., Nature In press]. Finally, our single cell analyses performed in part 1 also revealed that cholangiocytes express extremely higher level of ACE2 the main receptor of SARS-CoV2 [Sungnak et al., Nat. Medicine 2020]. Thus, we developed a new research project on Covid19 using our cholangiocytes organoids as a platform to study SARS-Cov2 infection. Using our model system, we identified mechanisms controlling the virus receptor expression on key cell types and also uncover that drug currently used against liver disease could modulate ACE2 expression [Brevini et al., Nature 2023].
The last part of the program explored the interest of cholangiocyte organoids for cell-based therapies against cholangiopathies. For that, we bioengineered an artificial common bile duct and showed that this construct can be engrafted successfully in mouse model [Sampaziotis et al., Nat. Medicine 2017]. We then also show that cholangiocytes could engraft and repair the intra-hepatic biliary epithelium in mouse model for cholangiopathy and in ex vivo human perfused liver [Sampaziotis et al., Science 2021].
While we collected primary cells for transcriptomic analyses, we also derived Cholangiocytes organoids from the same samples. The cells collected were then grown in 3D conditions using culture media specially developed for this program [Samapaziotis et al., Nat. Medicine 2017]. The resulting organoids were characterised as described [Tysoe et al., Nat. Protocols 2019] and their transcriptome compared to those of primary tissue [Sampaziotis et al., Nat Protocols 2017]. These experiments show that primary cholangiocyte organoids maintain their functional activities for bile acid and drug transports. However, organoids derived from the different region of the biliary tree lose their regionalisation [Sampaziotis et al., Science 2021]. Regionalisation could be re-established in organoids by adding in our culture conditions niche factors such as bile acids. Thus, our results have established that cholangiocytes can adopt different regional state in function of their microenvironment. This plasticity could explain in part the regionality of cholangiopathies. These results were also used to define the nomenclature of cholangiocytes organoids in the hepatology field [Marsee et al., Cell Stem Cell 2021].
We then explored the interest of cholangiocyte organoids to model cholangiopathies in vitro. Fort that, we showed that monogenetic disorders such as Alagille Syndrome can be modelled in vitro by blocking Notch signalling [Sampaziotis et al., Nat. Biotech 2015] or by genome editing using protocol developed for hiPSCs [Bertero et al., Development 2016]. We also validate the interest of intrahepatic cholangiocytes organoids (ICOs) to study idiopathic cholangiopathies. Finally, we have established a platform to study ductular reaction (DR) a process which is commonly observed during the progression of chronic liver diseases such as Metabolic dysfunction-associated fatty liver disease (MASLD). DR involves the proliferation of cholangiocytes and a profound remodelling of the biliary tree resulting in further disease progression. However, it has been suggested that DR could also represent a regenerative process aiming to repair the liver. In sum, we have shown that ICOs grown in vitro can be used to model DR and the associated regenerative processes [Gribben et al., Nature In press]. Finally, our single cell analyses performed in part 1 also revealed that cholangiocytes express extremely higher level of ACE2 the main receptor of SARS-CoV2 [Sungnak et al., Nat. Medicine 2020]. Thus, we developed a new research project on Covid19 using our cholangiocytes organoids as a platform to study SARS-Cov2 infection. Using our model system, we identified mechanisms controlling the virus receptor expression on key cell types and also uncover that drug currently used against liver disease could modulate ACE2 expression [Brevini et al., Nature 2023].
The last part of the program explored the interest of cholangiocyte organoids for cell-based therapies against cholangiopathies. For that, we bioengineered an artificial common bile duct and showed that this construct can be engrafted successfully in mouse model [Sampaziotis et al., Nat. Medicine 2017]. We then also show that cholangiocytes could engraft and repair the intra-hepatic biliary epithelium in mouse model for cholangiopathy and in ex vivo human perfused liver [Sampaziotis et al., Science 2021].
Each part of the project has resulted in the development of new technology. Using our single cell transriptomic data, we have identified the transcriptional network characterising not only cholangiocytes but also hepatocytes. This knowledge was used to identify transcription factors for forward programming applications. Accordingly, we identified HNF1, FOXA3, HNF6 and RORC to be essential in the specification of hepatocytes. These factors were then used in combination with the OptiOx system to generate FoP-Hepatocytes in vitro.
We have also developed culture conditions to derive and to grow cholangiocytes organoids from the extra- and intra-hepatic biliary tree. The resulting culture conditions can be used to expend cholangioctes for disease modelling and cell-based therapy applications. This work results into new intellectual property which was used to create the biotech company Bilitech (http://bilitech.co.uk/(opens in new window)).
In parallel, we have exploited the unique properties of cholangiocytes organoids to model disease including SARS-CoV2 infection. Using this platform, we identified new candidate drugs against COVID19. We also identify also a number of potential drug targets for monitoring progression of liver.
Finally, we established the first proof of concpet that cell-based therapies against cholangiopathies using biliary organoids could be feasible. This activity resulted in a new bioengineering method to generate artificial bile duct. Finally, we demonstrated for the first time that ex-vivo perfused human liver could provide a unique platform to test strategy for cell-based therapy.
We have also developed culture conditions to derive and to grow cholangiocytes organoids from the extra- and intra-hepatic biliary tree. The resulting culture conditions can be used to expend cholangioctes for disease modelling and cell-based therapy applications. This work results into new intellectual property which was used to create the biotech company Bilitech (http://bilitech.co.uk/(opens in new window)).
In parallel, we have exploited the unique properties of cholangiocytes organoids to model disease including SARS-CoV2 infection. Using this platform, we identified new candidate drugs against COVID19. We also identify also a number of potential drug targets for monitoring progression of liver.
Finally, we established the first proof of concpet that cell-based therapies against cholangiopathies using biliary organoids could be feasible. This activity resulted in a new bioengineering method to generate artificial bile duct. Finally, we demonstrated for the first time that ex-vivo perfused human liver could provide a unique platform to test strategy for cell-based therapy.