Cholangiocyte organoids from human bile retain a local phenotype and can repopulate bile ducts in vitro

Abstract

The well‐established 3D organoid culture method enabled efficient expansion of cholangiocyte‐like cells from intrahepatic (IHBD) and extrahepatic bile duct (EHBD) tissue biopsies. The extensive expansion capacity of these organoids enables various applications, from cholangiocyte disease modelling to bile duct tissue engineering. Recent research demonstrated the feasibility of culturing cholangiocyte organoids from bile, which was minimal‐invasive collected via endoscopic retrograde pancreaticography (ERCP). However, a detailed analysis of these bile cholangiocyte organoids (BCOs) and the cellular region of origin was not yet demonstrated. In this study, we characterize BCOs and mirror them to the already established organoids initiated from IHBD‐ and EHBD‐tissue. We demonstrate successful organoid‐initiation from extrahepatic bile collected from gallbladder after resection and by ERCP or percutaneous transhepatic cholangiopathy from a variety of patients. BCOs initiated from these three sources of bile all show features similar to in vivo cholangiocytes. The regional‐specific characteristics of the BCOs are reflected by the exclusive expression of regional common bile duct genes ( HOXB2 and HOXB3 ) by ERCP‐derived BCOs and gallbladder‐derived BCOs expressing gallbladder‐specific genes. Moreover, BCOs have limited hepatocyte‐fate differentiation potential compared to intrahepatic cholangiocyte organoids. These results indicate that organoid‐initiating cells in bile are likely of local (extrahepatic) origin and are not of intrahepatic origin. Regarding the functionality of organoid initiating cells in bile, we demonstrate that BCOs efficiently repopulate decellularized EHBD scaffolds and restore the monolayer of cholangiocyte‐like cells in vitro. Bile samples obtained through minimally invasive procedures provide a safe and effective alternative source of cholangiocyte organoids. The shedding of (organoid‐initiating) cholangiocytes in bile provides a convenient source of organoids for regenerative medicine.

Scalable Production of Size-Controlled Cholangiocyte and Cholangiocarcinoma Organoids within Liver Extracellular Matrix-Containing Microcapsules

“…Of note, the thickness of the cell layers appeared thicker in the encapsulated ICO, which could be due to the columnar polarization of the cholangiocytes inside the microcapsules. Similar phenotypes were seen for cholangiocyte organoids grown on ductal ECM when cell growth was limited by space (e.g., confluent cell layers) [ 38 , 39 ].…”

Design by Nature: Emerging Applications of Native Liver Extracellular Matrix for Cholangiocyte Organoid-Based Regenerative Medicine

“…Cholangiocyte organoids were established from gallbladder tissue (gallbladder cholangiocyte organoids; GCO) [28], and from the cholangiocytes' inner lining the extrahepatic bile duct (EBD, extrahepatic cholangiocyte organoids; ECO) [27,29]. Organoids were also initiated from the circulating EPCAM positive cells in fresh bile samples (bile-derived cholangiocyte organoids, BCO) [30]. These organoids all share similar phenotypic features when cultured, as they all grow in similar spherical structures with comparable proliferation rates [27,30,31].…”

“…Organoids were also initiated from the circulating EPCAM positive cells in fresh bile samples (bile-derived cholangiocyte organoids, BCO) [30]. These organoids all share similar phenotypic features when cultured, as they all grow in similar spherical structures with comparable proliferation rates [27,30,31]. They express similar progenitor and mature cholangiocyte markers, but are uniquely related to their original tissue of origin and show subtle differences.…”

“…Cholangiocyte organoid-derived cells are a promising source of cells for repopulation of the biliary tree. We recently showed that cells derived from ECO and BCO were capable or repopulating small discs (Ø3 mm) of decellularized extrahepatic bile duct (EBD) tissue [30,155]. They self-organized into polarized monolayers resembling biliary epithelium (Figure 4).…”

Organoids and regenerative hepatology

“…[105,106] Decellularized human liver scaffolds can then be repopulated with functional liver cells. [105,106] Although cholangiocyte organoids [107,108] have also been used to repopulate small flat patches of decellularized extrahepatic ducts, the potential of this approach for tissue repair or regenerative medicine has not been demonstrated. Challenges related to vascularization and upscaling this technology to human size still remain to be addressed.…”