Patch grafting of expanded biliary tree stem cell organoids to rescue bile duct injury

Biliary tree stem cells (BTSCs) have the potential of hepatobiliary-pancreatic differentiation and are ideal for repairing bile duct damage. Previously, we established a BTSC Expansion Hydrogel (BEX-gel) system for expansion of BTSC organoids, and developed a patch grafting strategy to deliver BTSC organoids for liver injury and diabetes treatment. In this study, we focused on the ability of the expanded BTSC organoids to give rise to functional cholangiocyte organoids, as well as their capacity of rescuing bile duct injury. A cholangiocyte differentiation system, composed of matrix plus serum-free Kubota's Medium supplemented with FGF2, VEGF165, HGF, TGF-β1, Jagged-1, Retinoid Acid (RA), Forskolin and with Cu (2+) and Ca (2+) to induce the expanded BTSC organoids to form cholangiocyte organoids within 7 days. RNA-sequencing was used to compare BTSC organoids, BTSC-derived branching cholangiocyte organoids (BTSC-ChOs), and mature murine cholangiocytes (mCHOs). RNA-seq showed that the gene expression of cholangiocyte markers Sctr, Ae2, Ggt1 and Tgr5 was significantly upregulated in BTSC-ChOs. Functional assays, including the Rhodamine-123 transport and intracellular calcium levels indicated that BTSC-ChOs acquired similar properties to those of mCHOs. Transplantation of BTSC organoids via patch grafting strategy into animals with bile duct injuries due to a DDC-diet and MDA intraperitoneal injection resulted in in vivo maturation into functional cholangiocytes for bile duct repair. Further analysis of primary bile acid expression levels before and after treatment confirmed that Tauro-β-muricholic Acid (TβMCA) and Taurocholic acid (TCA) are highly sensitive to the degree of bile duct repair by transplanted cells, therefore, provide ideal clinical monitoring parameters for the treatment of bile duct injuries. This study presents the expanded BTSC organoids as an ideal source of cells for therapeutic treatment of diseases associated with bile duct injuries.