Single‐Cell, Single‐Nucleus, and Spatial RNA Sequencing of the Human Liver Identifies Cholangiocyte and Mesenchymal Heterogeneity

Abstract

The critical functions of the human liver are coordinated through the interactions of hepatic parenchymal and non‐parenchymal cells. Recent advances in single‐cell transcriptional approaches have enabled an examination of the human liver with unprecedented resolution. However, dissociation‐related cell perturbation can limit the ability to fully capture the human liver’s parenchymal cell fraction, which limits the ability to comprehensively profile this organ. Here, we report the transcriptional landscape of 73,295 cells from the human liver using matched single‐cell RNA sequencing (scRNA‐seq) and single‐nucleus RNA sequencing (snRNA‐seq). The addition of snRNA‐seq enabled the characterization of interzonal hepatocytes at a single‐cell resolution, revealed the presence of rare subtypes of liver mesenchymal cells, and facilitated the detection of cholangiocyte progenitors that had only been observed during in vitro differentiation experiments. However, T and B lymphocytes and natural killer cells were only distinguishable using scRNA‐seq, highlighting the importance of applying both technologies to obtain a complete map of tissue‐resident cell types. We validated the distinct spatial distribution of the hepatocyte, cholangiocyte, and mesenchymal cell populations by an independent spatial transcriptomics data set and immunohistochemistry. Conclusion: Our study provides a systematic comparison of the transcriptomes captured by scRNA‐seq and snRNA‐seq and delivers a high‐resolution map of the parenchymal cell populations in the healthy human liver.

Single Nucleus RNA Sequencing of Pre-Malignant Liver Reveals Disease-Associated Hepatocyte State with HCC Prognostic Potential

“…Recent single cell studies unveiled the transcriptional heterogeneity of hepatocyte zones in both mouse (Halpern et al, 2017; Nault et al, 2021) and human (Aizarani et al, 2019; Payen et al, 2021) liver in high detail. Consistent with previous findings (Aizarani et al, 2019; Andrews et al, 2021; Halpern et al, 2017; Payen et al, 2021), hepatocytes in our dataset were found in a gradient between two highly distinctive states. One representing zone 1 (periportal) hepatocytes, which was characterized by high expression of genes such as Sds, Hal and Gls2, and a second representing zone 3 (pericentral) hepatocytes, which expressed Glul, Slac1a2 and Lgr5.…”

“…Hepatocytes constitute approximately 60% of the liver by cell number (Stanger, 2015), however due to their sensitivity to tissue dissociation, have not been well-represented in current single cell liver disease datasets. Although tissue dissociation approaches better suited to improve hepatocyte representation have been reported, they have only been applied to study healthy liver (Andrews et al, 2021; MacParland et al, 2018; Payen et al, 2021). Moreover, solid tissue dissociation for scRNA-seq introduces cell representation biases and de novo transcriptional stress responses, which may mask the underlying biological state under study (Denisenko et al, 2020).…”

Single cell profiling of functionally cured Chronic Hepatitis B patients reveals the emergence of activated innate and an altered adaptive immune response in the intra-hepatic environment

“…We employed Harmony-based integration (19) to eliminate variations arising due to batch differences and sequencing platforms and observed clear hierarchical clustering of each cell type and representation from each patient in the individual cell clusters. Cell type annotation of Leiden clusters was based on lineage-specific markers adapted from earlier scRNA-seq studies profiling the liver (20–22) (Fig. 1C and 1F), such as T cells ( CD3E, CD3D, IL7R, CD8A, CD8B, TRAC ), NK cells ( NCAM1, GNLY, NKG7, TRDC, PRF1 ), myeloid cells ( CD14, CSF1R, FCGR3A, C1QA, C1QB, IDO1, CLEC10A ), B cells ( MS4A1, MZB1, CD79A, JCHAIN ), stellate cells ( ACTA2, RGS5, COL1A1 ), endothelial cells ( PECAM1, RELN, ENG, FSCN1, CAVIN2 ), and hepatocytes ( ALB, CYP3A4 ).…”

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

“…Different variants of snRNA-seq have been applied in studying various human tissue types. 58 , 59 , 60 , 61 In a recent study by Andrews et al, 62 the transcriptomic landscape of the human liver was studied by matched scRNA-seq and snRNA-seq. The use of snRNA-seq revealed the presence of rare subtypes of liver mesenchymal cells, while lymphocytes were only distinguishable by scRNA-seq.…”