Single‐Cell Transcriptomic Analysis Reveals a Hepatic Stellate Cell–Activation Roadmap and Myofibroblast Origin During Liver Fibrosis in Mice

Abstract

BaCKgRoUND aND aIMS: HSCs and portal fibroblasts (PFs) are the major sources of collagen-producing myofibroblasts during liver fibrosis, depending on different etiologies. However, the mechanisms by which their dynamic gene expression directs the transition from the quiescent to the activated state-as well as their contributions to fibrotic myofibroblasts-remain unclear. Here, we analyze the activation of HSCs and PFs in CCL 4 -induced and bile duct ligation-induced fibrosis mouse models, using single-cell RNA sequencing and lineage tracing. appRoaCH aND ReSUltS:We demonstrate that HSCs, rather than PFs, undergo dramatic transcriptomic changes, with the sequential activation of inflammatory, migrative, and extracellular matrix-producing programs. The data also reveal that HSCs are the exclusive source of myofibroblasts in CCL 4 -treated liver, while PFs are the major source of myofibroblasts in early cholestatic liver fibrosis. Single-cell and lineage-tracing analysis also uncovers differential geneexpression features between HSCs and PFs; for example, nitric oxide receptor soluble guanylate cyclase is exclusively expressed in HSCs, but not in PFs. The soluble guanylate cyclase stimulator Riociguat potently reduced liver fibrosis in CCL 4 -treated livers but showed no therapeutic efficacy in bile duct ligation livers. CoNClUSIoNS:This study provides a transcriptional roadmap for the activation of HSCs during liver fibrosis and yields comprehensive evidence that the differential transcriptomic features of HSCs and PFs, along with their relative contributions to liver fibrosis of different etiologies, should be considered in developing effective antifibrotic therapeutic strategies. (Hepatology 2021;74:2774-2790.

Single‐cell transcriptomics reveals conserved cell identities and fibrogenic phenotypes in zebrafish and human liver

“…In contrast to the ECs, the majority of human and zebrafish HSCs clustered together (zhHSC) (Figure 4B ; Figure S5A ). Expression of human HSC marker genes α‐actin 2 ( ACTA2 ) , collagen type I alpha 1 chain ( COL1A1 ) , collagen type I alpha 2 chain ( COL1A2 ) , SPARC , and COLEC11 [ 10 , 37 ] was observed in over 75% of all human HSCs (Figure 4C ). Of these genes, only SPARC and COLEC11 were highly captured in zebrafish HSCs, while known zebrafish HSC marker, HAND2 , and markers determined from our zebrafish scRNA‐seq data set, angiopoietin‐like 6 ( ANGPTL6 ) and STEAP4 , were poorly captured in human HSCs (Figure 4C ).…”

“…Of these genes, only SPARC and COLEC11 were highly captured in zebrafish HSCs, while known zebrafish HSC marker, HAND2 , and markers determined from our zebrafish scRNA‐seq data set, angiopoietin‐like 6 ( ANGPTL6 ) and STEAP4 , were poorly captured in human HSCs (Figure 4C ). Commonly used protein markers for human HSCs, including glial fibrillary acidic protein (GFAP), platelet‐dervied growth factor recepor beta (PDGFRB), lecithin retinol acyltransferase (LRAT), desmin (DES), [ 37 , 38 ] were poorly captured by scRNA‐seq in both human and zebrafish HSCs (Figure 4C ). These findings indicate that traditional markers of human and zebrafish HSCs at the transcriptional level show lower levels of conservation.…”

“…To determine if induction of Nrp1 in cholangiocytes was only observed in Mpi depletion or was more broadly found in other models of biliary fibrosis, we queried published scRNA‐seq data on livers from mice with bile duct ligation (BDL), [ 37 ] a known model of biliary fibrosis, compared to controls and CCl 4 ‐induced liver fibrosis conditions. We analyzed the SRY‐box transcription factor 9 ( Sox9 )+ cholangiocyte population found in this data set and found that Nrp1 was significantly up‐regulated in livers from BDL mice when compared to control and CCl 4 livers (Figure 6E ).…”

Single-Cell Transcriptomics of Liver Cancer: Hype or Insights?

“… 52 In fact, there are interesting studies describing single-cell investigations related to hepatic stellate cells, which are critical to the pathogenesis of liver fibrosis 53 , 54 and intrahepatic cholangiocarcinoma. 55 , 56 , 57 Together with the aforementioned investigations in HCC, they represent the major parts in the hepatocarcinogenesis spectrum of primary liver cancer.…”

Understanding the cellular interactome of non-alcoholic fatty liver disease

“…PFs are a key myofibroblast precursor in cholestatic liver injury, 133 and populations of PFs have been shown to expand in murine models of biliary injury, expressing high levels of fibrillar collagen at single-cell resolution. 134 These cells are distinct from HSCs with no vitamin A storage and express unique markers such as THY1, fibulin (FBLN)1, FBLN2, MFAP4 (microfibril associated protein 4) and GAS6 (growth arrest specific 6). [135][136][137]…”