Achieving scalable expansion of therapeutic porcine hepatocytes in vivo through serial transplantation

The clinical application of hepatocyte transplantation has been significantly hindered by the scarcity of primary hepatocytes and the functional immaturity of in vitro-produced hepatocytes. By performing serial allogeneic hepatocyte transplantation in CRISPR/Cas9-mediated Fah-knockout pigs, we successfully achieved large-scale expansion of hepatocytes while maintaining their authentic biological characteristics. Particularly, the established model enables sustained in vivo liver reconstruction, concurrently ameliorating hepatic fibrosis and demonstrating functional microenvironmental remodeling. Moreover, through comprehensive single-cell transcriptomic profiling of 52 418 hepatocytes across transplant generations (F0-F2), we discovered that the cellular composition of these transplanted hepatocytes is similar to that of wild-type hepatocytes. The regenerated liver exhibits all six major hepatic cell types identical to the wild-type counterparts, with the characteristic lobular zonation patterns well preserved. Our research provides valuable insights into the large-scale expansion of physiologically functional hepatocytes in vivo without compromising their biological properties. This finding holds great promise for advancing the clinical application of human hepatocyte transplantation, potentially offering more effective treatment options for patients with liver diseases.