Cell Transplantation Promising for Treatment of Biliary Diseases

Biliary disease is a term used to describe diseases that affect the biliary system, which can result in inflammation, fibrosis, bile duct destruction, and eventually liver failure. It also frequently leads to secondary infections and chronic irritation, such as bile duct stones, which can lead to malignant tumors. There is currently no cure for biliary tract diseases other than liver transplantation, so new modalities of treatment are urgently needed.

Biliary epithelial cells (BECs) are a heterogeneous population that has been shown to have dual potential. When liver regeneration is severely impaired in mice, BECs can develop into hepatocytes or bile duct cells. This bipotential cell population can be isolated, expanded, and transplanted to repopulate the damaged liver soft tissue and differentiate into mature hepatocytes in order to restore liver function when endogenous regenerative mechanisms are exhausted. It has been demonstrated that both transplanted bile duct cells and hepatocyte-derived cells produce biliary regeneration in mice, which fully demonstrates the therapeutic potential of BECs in liver diseases. However, the use of human BECs (hBECs) in the clinical setting remains limited.

On March 3, 2022, Stuart J. Forbes' team from the University of Edinburgh, UK, published an article online in Cell Stem Cell titled "Human biliary epithelial cells from discarded donor livers rescue bile duct structure and function in a mouse model of biliary disease", offering new hope for the application of transplant regeneration of clinical hBECs. The researchers isolated and expanded hBECs from discarded livers and transplanted them into a mouse model of biliary disease. hBECs regenerated and repaired damaged bile ducts, reduced liver fibrosis, and decreased mortality. This study provides a novel therapeutic approach for biliary tract diseases.

To identify and characterize hBECs with therapeutic potential, researchers isolated and sorted candidate hBEC clusters from liver tissue. The classification strategy used was the sorting method previously used to isolate BECs from mice with dual potential, and two clusters of candidate hBECs were obtained: CD45-/CD31-/EpCAM+/CD24+/CD133- (referred to as CD133-) and CD45-/CD31-/EpCAM+/CD24+/CD133+ (referred to as CD133+).

Subsequently, the researchers characterized the full range of hBECs isolated from adipose and healthy livers.

• Whole transcriptome analysis: genes related to proliferation were up-regulated and genes related to the regulation of immune system processes (e.g., MUC1, MUC5B), inflammatory response-related genes (e.g., IL1LR1, GPX4, ADCY5) and extracellular matrix tissue-related genes (LAMB3, MMP1) were down-regulated, suggesting that CD133+ hBECs may have greater regenerative capacity.
• The cellular regenerative potential of CD133+ hBECs was assessed: genes significantly upregulated in steatosis liver-derived CD133+ hBECs included those related to cell proliferation, metabolism, and extracellular matrix organization; genes related to inflammatory responses and chemokine-mediated signaling were significantly downregulated, indicating that the regenerative potential of hBECs was increased under steatosis conditions.
• The clonogenic ability of CD133- and CD133+ hBECs was investigated: compared to CD133- hBECs, CD133+ hBECs showed significantly higher clonogenic efficiency, forming clones that underwent continuous transmission and survived for more than 15 weeks while retaining the normal diploid karyotype.
• D133+ hBECs can grow into organoids and express bile duct cells and progenitor cell markers (e.g., K19, SOX9, EpCAM, STEM121, and LGR5); CD133+ hBECs are also able to differentiate into a hepatocyte lineage, displaying distinct morphological and transcriptional features, suggesting similar differentiation potential between human and mouse BECs.

Next, the researchers transplanted the amplified hBECs into a mouse model of immune-compromised biliary disease to assess the regenerative capacity of the CD133+ hBECs population in vivo. Follow-up analysis revealed that mice transplanted with CD133+ hBECs improved in several aspects compared to control mice: mice had fewer intrahepatic lesions, less hepatosplenomegaly, lower bilirubin levels, significantly higher survival rates, and significantly less fibrosis.

Further, the researchers explored the biological mechanisms by which hBEC transplantation ameliorates the disease phenotype. hBEC transplantation restored the anatomy of the intrahepatic biliary tract and reduced parenchymal damage in the polypoid-infiltrating nucleus. The results showed that mice in the PBS-treated group developed bile duct occlusion, stenosis, and dilated gallbladder and CBD. In contrast, hBEC transplanted mice showed a significant decrease in CBD and gallbladder diameter and an increase in bile duct, indicating that transplantation improved stenosis in the extrahepatic region of the biliary tract.

Finally, the authors also developed an isolation and culture process that is compliant with current GMP regulations. The process uses automated steps and GMP-compliant reagents for liver isolation followed by clinical-grade magnetic bead sorting.

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