Ever since the discovery of ways to culture epithelial organoids, their application for organ regeneration or tissue engineering has been anticipated. Now an article published by Sampaziotis et al1 in Science titled “Cholangiocyte organoids can repair bile ducts after transplantation in the human liver” delivers the first proof of principle. This study shows repair of the biliary tree, both in a murine model and in discarded human liver grafts. The authors demonstrate for the first time a possible treatment to repair the biliary tree during ex vivo normothermic machine perfusion (NMP) using organoids.
In the field of liver transplantation, grafts donated after circulatory death (DCD) are prone to posttransplantation complications.2 Especially, the risk of nonanastomotic strictures (NAS) is high. In “standard” DCD grafts, the risk of developing NAS is approximately 4 times higher than in grafts donated after brain death.2 The (functional) warm ischemia causes injury in the peribiliary glands of the bile duct. The peribiliary glands are assumed to be a niche of biliary stem cells and therefore critical for the regeneration of the biliary tree after injury.3 When the peribiliary glands are injured, the risk of developing NAS increases significantly.3 NAS remains a challenging clinical problem with a high risk of retransplantation.
Organ perfusion techniques such as (dual)-hypothermic machine perfusion improve preservation of DCD grafts and protect peribiliary glands. In a recent study, (dual)-hypothermic machine perfusion prevented NAS formation in 67%; however, still 6% of the grafts that were treated with dual-hypothermic machine perfusion developed NAS.4 This study was performed with strict upfront eligibility criteria of the DCD donors. Presumably, the NAS incidence will increase under less stringent conditions. In current practice, extended DCD grafts are usually evaluated with ex vivo NMP. NMP cannot prevent NAS formation; however, with NMP the quality of the biliary tree can be evaluated.5,6 Physiologically, cholangiocytes excrete bicarbonate in bile and extract amino acids and glucose out of the bile. In injured bile ducts, these processes are impaired. By analyzing the pH of bile, bicarbonate levels, and glucose levels during ex vivo NMP, cholangiocyte function and biliary integrity can be assessed.5,6
Sampaziotis et al1 showed a way to repair the biliary tree with cholangiocyte organoids before liver transplantation. Previously the team of Ludovic Vallier at the University of Cambridge, United Kingdom, reported on a new method for the isolation and propagation of human cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids and tested their regenerative medicine applications.7 Now, these cholangiocyte organoids have for the first time been transplanted into discarded human liver grafts. The organoids (approximately 10 million cells) were infused directly into the peripheral branches of the bile duct. This retrograde administration against the direction of the normal bile flow facilitated the engraftment into peripheral small branches of the bile ducts rather than being washed out. Using a red fluorescent protein label, the infused organoids could be tracked upon engraftment. Approximately 60% of all cholangiocytes in the treated segments were red fluorescent protein positive after 100 hours of NMP. These organoids provided a significant functional benefit. The total volume of bile produced by liver segments receiving organoids was significantly higher compared with graft segments treated with carrier alone. Also, the biliary pH was significantly increased in the treatment group. Whether engraftment of organoids is effective to prevent NAS was not demonstrated, as these liver segments were not actually transplanted. Also, the study does not report whether organoid cells engraft permanently in the peribiliary glands, which may be particularly relevant for the prevention of NAS.
Organoids derived from ductal tissue located inside or outside the liver share many characteristics, including the expression of cholangiocyte-, stem cell-, and progenitor markers.8 However, RNA sequencing revealed that organoids conserve some regional-specific properties and differentiation potential corresponding to their location of origin.8,9 Despite these differences, cholangiocyte organoids were shown to have a high degree of plasticity, adapting to the local microenvironment along the biliary tree when engrafted. However, the plasticity of cholangiocyte organoids is not without limits as they will not restore the hepatocellular compartment of the liver. For graft repair, ideally, autologous cells of the recipient have been used to diminish the risk of alloimmune responses. This poses the question on the ideal source of autologous cells to culture and biobank cholangiocyte organoids before liver transplantation. It has been shown that autologous cholangiocyte organoids can be derived from different sources, including liver biopsies or even from bile.9,10 Obtaining bile could be relatively noninvasively collected by percutaneous drainage of the gallbladder or via endoscopic retrograde cholangiopancreatography in outpatient clinics.
The best timing for cholangiocyte organoid treatment remains an additional open question. Treating already established NAS bears the risk of being ineffective, as structural changes to the biliary tree may be irreversible. Scar tissue around the bile ducts which is formed in NAS, might also prevent engraftment of cholangiocyte organoids. Transplanted cells need both space and a supportive environment to engraft. Hence, infusing the organoid cells directly after the injury has occurred (due to prolonged warm or cold ischemia) might be optimal timing. Thus, ex vivo NMP as in Sampaziotis’s work is certainly 1 option. Alternatively, cholangiocyte organoids could be infused into the bile duct after reperfusion and before the biliary anastomosis.
In summary, Sampaziotis et al are the first showing the regenerative potential of organoids in human liver grafts. This work provides a therapeutic stem cell strategy to repair ischemic bile ducts, potentially preventing posttransplantation NAS and providing a prospect for organoid-based therapies for other cholangiopathies.
1. Sampaziotis F, Muraro D, Tysoe OC, et al. Cholangiocyte organoids can repair bile ducts after transplantation in the human liver. Science. 2021;371:839–846.
2. Kalisvaart M, de Haan JE, Polak WG, et al. Comparison of postoperative outcomes between donation after circulatory death and donation after brain death liver transplantation using the comprehensive complication index. Ann Surg. 2017;266:772–778.
3. op den Dries S, Westerkamp AC, Karimian N, et al. Injury to peribiliary glands and vascular plexus before liver transplantation predicts formation of non-anastomotic biliary strictures. J Hepatol. 2014;60:1172–1179.
4. van Rijn R, Schurink IJ, de Vries Y, et al.; DHOPE-DCD Trial Investigators. Hypothermic machine perfusion in liver transplantation—A Randomized Trial. N Engl J Med. 2021; 384:1391–1401.
5. van Leeuwen OB, de Vries Y, Fujiyoshi M, et al. Transplantation of high-risk donor livers after ex situ resuscitation and assessment using combined hypo- and normothermic machine perfusion: a Prospective Clinical Trial. Ann Surg. 2019;270:906–914.
6. Watson CJE, Kosmoliaptsis V, Pley C, et al. Observations on the ex situ perfusion of livers for transplantation. Am J Transplant. 2018;18:2005–2020.
7. Sampaziotis F, Justin AW, Tysoe OC, et al. Reconstruction of the mouse extrahepatic biliary tree using primary human extrahepatic cholangiocyte organoids. Nat Med. 2017;23:954–963.
8. Rimland CA, Tilson SG, Morell CM, et al. Regional differences in human biliary tissues and corresponding in vitro-derived organoids. Hepatology. 2021;73:247–267.
9. Verstegen MMA, Roos FJM, Burka K, et al. Human extrahepatic and intrahepatic cholangiocyte organoids show region-specific differentiation potential and model cystic fibrosis-related bile duct disease. Sci Rep. 2020;10:21900.
10. Roos FJM, Verstegen MMA, Muñoz Albarinos L, et al. Human bile contains cholangiocyte organoid-initiating cells which expand as functional cholangiocytes in non-canonical Wnt stimulating conditions. Front Cell Dev Biol. 2020;8:630492.