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The liver is the largest solid organ in the body and is critical for metabolic and immune functions. However, little is known about the cells that make up the human liver and its immune microenvironment. Here we report a map of the cellular landscape of the human liver using single-cell RNA sequencing. We provide the transcriptional profiles of 8444 parenchymal and non-parenchymal cells obtained from the fractionation of fresh hepatic tissue from five human livers. Using gene expression patterns, flow cytometry, and immunohistochemical examinations, we identify 20 discrete cell populations of hepatocytes, endothelial cells, cholangiocytes, hepatic stellate cells, B cells, conventional and non-conventional T cells, NK-like cells, and distinct intrahepatic monocyte/macrophage populations. Together, our study presents a comprehensive view of the human liver at single-cell resolution that outlines the characteristics of resident cells in the liver, and in particular provides a map of the human hepatic immune microenvironment., The development of single cell RNA sequencing technologies has been instrumental in advancing our understanding of tissue biology. Here, MacParland et al. performed single cell RNA sequencing of human liver samples, and identify distinct populations of intrahepatic macrophages that may play specific roles in liver disease.

Organoid technology holds great promise for regenerative medicine but has not yet been applied to humans. Here, we address this challenge in the context of cholangiocyte organoids and cholangiopathies, which represent a leading indication for liver transplantation. Using single-cell RNA sequencing we show that primary human cholangiocytes display transcriptional diversity which is lost in organoid culture. However, cholangiocyte organoids remain plastic and resume their in vivo signatures when transplanted back in the biliary tree. We then utilize a new model of cell engraftment in human livers undergoing ex vivo normothermic perfusion to demonstrate that this property allows extrahepatic organoids to repair human intrahepatic ducts after transplantation. Our results provide proof-of-principle that cholangiocyte organoids can be used to repair human biliary epithelium.

Transdifferentiation is a complete and stable change in cell identity that serves as an alternative to stem-cell-mediated organ regeneration. In adult mammals, findings of transdifferentiation have been limited to the replenishment of cells lost from preexisting structures, in the presence of a fully developed scaffold and niche 1 . Here we show that transdifferentiation of hepatocytes in the mouse liver can build a structure that failed to form in development-the biliary system in a mouse model that mimics the hepatic phenotype of human Alagille syndrome (ALGS) 2 . In these mice, hepatocytes convert into mature cholangiocytes and form bile ducts that are effective in draining bile and persist after the cholestatic liver injury is reversed, consistent with transdifferentiation. These findings redefine hepatocyte plasticity, which appeared to be limited to metaplasia, that is, incomplete and transient biliary differentiation as an adaptation to cell injury, based on previous studies in mice with a fully developed biliary system3-6. In contrast to bile duct development7-9, we show that de novo bile duct formation by hepatocyte transdifferentiation is independent of NOTCH signalling. We identify TGFβ signalling as the driver of this compensatory mechanism and show that it is active in some patients with ALGS. Furthermore, we show that TGFβ signalling can be targeted to enhance the formation of the biliary system from hepatocytes, and that the transdifferentiation-inducing signals and remodelling capacity of the bile-duct-deficient liver can be harnessed with transplanted hepatocytes. Our results define the regenerative potential of mammalian transdifferentiation and reveal opportunities for the treatment of ALGS and other cholestatic liver diseases.