Portal fibroblasts: A renewable source of liver myofibroblasts
Authors: Steven P. O’Hara, Nicholas F. LaRusso
Journal: Hepatology (2022)

SEE ARTICLE ON PAGE 1360 Fibrosis leading to cirrhosis is the final common pathway of chronic hepatitis and cholestatic liver injury. Despite this confluence of multiple diseases into a common pathological process, distinct liver injuries initially exhibit different fibrotic patterns. For example, the initial distribution of fibrosis in cholestatic diseases, like primary biliary cholangitis and primary sclerosing cholangitis (PSC), is localized around portal tracts, whereas alcohol‐associated liver disease shows deposition of fibrous material in pericentral and perisinusoidal areas.[1] These distinct fibrotic patterns strongly suggest that cell‐specific injuries initiate different signaling pathways between spatially and perhaps phenotypically unique fibrogenic cell types. Indeed, the spatial distribution of hepatic fibrogenic cells was recognized decades ago, when hepatic mesenchymal cells, not associated with the sinusoids, were described. It was also noted that fibroblast‐like cells were present in the region immediately surrounding proliferating bile ducts in biliary cirrhosis.[2] The spatial localization of portal fibroblasts (PFs) made them attractive candidates as mediators of biliary fibrosis, and a model whereby PFs were first responders in biliary fibrosis, later to be supplanted by hepatic stellate cells (HSCs), was first proposed in 2002 by Kinnman and Housset.[3] Additionally, over a decade ago, this journal published an insightful review by Dranoff and Wells that discussed PFs in hepatic fibrogenesis, described gaps in knowledge regarding this enigmatic cell, and defined areas for further exploration.[4] In this issue of Hepatology, Lei et al. use state‐of‐the‐art technologies and bioinformatic analyses to further define the cellular composition of the portal mesenchyme, identify markers to differentiate between myofibroblast subpopulations, and define a role of PFs in mouse and human pathologies.[5] It is expected that this work will aid in the continued interest and promote an additional resurgence of studies focused on PFs and their contribution to liver disease. The mesenchymal cells of the liver are composed of sinusoidal HSCs and perivascular cells, including vascular smooth muscle cells (VSMCs) and fibroblasts found in the portal tracts and around the central vein. These mesenchymal cells are derived from the embryonic septum transversum mesenchyme (STM).[6] The STM induces expansion of the ventral foregut endoderm forming the liver bud. Cords of primitive hepatic cells grow into this mesenchyme forming hepatic sinusoid‐like structures; mesenchymal cells localized between the hepatic cells and developing liver sinusoidal endothelial cells will ultimately become the HSCs.[7] The nascent portal venous system, branches of veins throughout the developing liver bud, drives hepatoblast differentiation to form the cholangiocytes through ductal plate morphogenesis. Intrahepatic arterial branches tract along the nascent intrahepatic bile ducts extending from the central to peripheral areas of the developing liver, and the portal triad, i.e., portal vein, hepatic artery, and bile duct, is thus established. This portal triad is embedded in hepatic mesenchyme, yet the complete cellular composition of this mesenchyme and its contribution to liver fibrosis has remained ill‐defined. Myofibroblasts, matrix‐producing cells characterized by shared gene expression with smooth muscle cells, are effector cells of fibrosis in fibrotic diseases. HSCs, quiescent vitamin A–rich mesenchymal cells, were the first cells to be identified as a source of myofibroblasts during liver injury.[8] Following refinement of techniques for HSC isolation, many studies focused on HSCs, which are now recognized as the major source of myofibroblasts in liver fibrosis. The authors of the work presented herein previously used outgrowths from fragments of the biliovascular tree of rat liver to demonstrate that portal mesenchymal cells, distinct from HSCs, generate highly proliferative, collagen type XV alpha 1‐positive myofibroblasts that have proangiogenic properties.[9] Here, Lei et al. extend their work to further define portal mesenchymal cellular diversity and identify surface markers used to isolate and examine the role of these cells in murine and human hepatic disease. To assess the cellular composition of the portal mesenchyme, the authors isolated single cell suspensions from the biliovascular trees from healthy mice. Through cell sorting, they enriched cells that were negative for lineage markers of cholangiocytes and endothelial and hematopoietic cells. Single‐cell RNA sequencing (RNAseq) was then performed on this enriched population of portal mesenchymal cells. Unsupervised clustering identified 16 cell clusters including mesothelial cells, likely from Glissonean pedicles, and four clusters of endothelial cells that likely escaped the selection process. Of the mesenchymal cell components, they identified five distinct clusters of both fibroblasts and VSMCs and one distinct cluster of HSCs. All five clusters of fibroblasts expressed extracellular matrix proteins, including several collagens. Yet, the clusters exhibited unique expression profiles leading to speculation of distinct cell fates during fibrogenesis. For example, fibroblast cluster 5 shared gene expression profiles with HSCs, suggesting that this cluster may be an intermediary between PFs and fully differentiated HSCs. Fibroblast clusters 3 and 4 were highlighted as potential progenitor cells based on measurements of single‐cell entropy, which approximates differentiation potential and plasticity of single cells based on signaling promiscuity.[10] Further analysis of the fibroblast, VSMC, and HSC clusters using trajectory and Gene Ontology‐term analyses as well as stem cell marker expression further supported that fibroblast clusters 3 and 4 exhibited multilineage potential and characteristics of mesenchymal stem cells. These clusters were designated as PFs with mesenchymal stem cell features (PMSCs). Hence, this series of thoughtful and sequentially logical, technically demanding experiments using sophisticated methodology revealed the complex cellular composition of the portal mesenchyme and identified clusters of cells that are the likely precursors to portal myofibroblasts. Surface markers were defined that allowed fluorescence‐activated cell sorting enrichment of PMSCs. Cell culture studies showed that these cells were highly clonogenic, expressed mesenchymal stem cell markers, and could undergo trilineage differentiation (a characteristic of mesenchymal stem cells). Moreover, when cultured on stiff substratum, but not when grown as spheroids, the cells exhibited portal myofibroblast characteristics. Thus, the authors convincingly demonstrate that their screening strategy and isolation procedure identified PMSCs that are likely precursors to portal myofibroblasts. The authors then performed bulk RNAseq to define the unique expression features of PMSCs contrasted to ultrapure HSCs. RNAseq was performed on PMSCs and HSCs from the quiescent state through myofibroblast transdifferentiation. A principal phenotypic feature observed throughout myofibroblast differentiation between these populations was that PMSCs were clonogenic in culture whereas HSCs were not. Moreover, the authors defined an expression signature that could demarcate the cells from the quiescent state through myofibroblastic differentiation. One transcript, slit guidance ligand 2 (Slit2), was identified as a prototypical gene of PMSCs. In vitro studies supported that PMSC‐derived Slit2 promotes endothelial cell neo‐vessel formation and HSC survival. Supporting this view, fluorescent in situ hybridization analyses showed that SLIT2+ PMSC‐myofibroblasts accumulated along neo‐vessels in the fibrotic septa of mouse models of liver disease and in human livers with cirrhosis. These structures were surrounded by SLIT2‐ myofibroblasts, likely derived from HSCs. Therefore, a functional role of portal myofibroblasts in fibrosis was proposed whereby expansion of PMSCs drive both neo‐vessel formation and robust accumulation of myofibroblasts derived from HSCs. The contributions of this work to the field are unique and significant. The authors identified markers to differentiate PMSCs, which have allowed them, and will allow others, to better determine the anatomical distribution and lineage of portal myofibroblasts in mouse and human liver samples. These markers can be used to identify the types of myofibroblasts present in fibrotic liver disease and how these myofibroblasts contribute to liver fibrogenesis over time and in response to different injuries. Given our own longstanding interest in the cholangiopathies, we are intrigued by the potential signaling networks among cholangiocytes and distinct populations of fibrogenic cells. Histologically, PSC is characterized by both periductal and bridging fibrosis potentially involving both PFs and HSCs sequentially or simultaneously.[11] Our ongoing work supports the notion that the response of cholangiocytes to injury in PSC is not uniform: that is, some cholangiocytes arrest in the cell cycle (i.e., become senescent) whereas others acquire a ductular reactive, proliferative phenotype. What accounts for this difference in stress response and its relevance to PSC pathogenesis remains unclear, yet it seems probable, if not likely, that senescent cholangiocytes restricted to the portal areas initially communicate directly with PFs, whereas the ductular reactive cells may more directly communicate with HSCs. Indeed, the portal localization of the characterized PMSCs make them attractive candidates as the initiators of biliary fibrosis, which may promote cellular signaling involving additional cell types including cholangiocytes, hepatocytes, VSMCs, and recruited and resident immune cells (Figure 1). Thus, the current work by Lei et al. sets the stage for further research that incorporates standardized markers to delineate the relative contributions of HSCs and PFs in fibrogenic processes of differing etiologies and provides tools for deciphering communication networks among distinct cell types in a variety of liver diseases.FIGURE 1: The authors have clarified the cellular composition of the portal mesenchyme and identified PMSCs. The markers and pathways identified herein advance our understanding of the signaling between myofibroblast subsets during fibrogenesis and establish reliable markers for continued investigation of this common pathogenic pathway of chronic liver disease. The current study allows reliable markers for further interrogation of the differences between hepatitis and cholestatic fibrogenic processes, periductal versus bridging fibrosis, and signaling networks between myofibroblast subsets and reactive cholangiocytes, endothelial cells, and resident and recruited immune cellsCONFLICT OF INTEREST Nothing to report.