Direct analysis of hepatic stellate cells with flow cytometry in specimens derived from the human liver

Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis. However, the methodology to directly assess the biology of primary HSCs in human liver specimens is yet to be established. In this study, we aimed to establish a robust methodology to analyse primary HSCs in human liver specimens with flow cytometry (FCM). We first applied FCM to HSCs directly isolated from liver tissues with Nycodenz density gradients. Then, we analysed HSCs in frozen/thawed liver perfusate samples and liver tissues. We also compared the phenotype of HSCs in primary biliary cholangitis (PBC) and those in healthy counterparts. We found that HSCs were substantially smaller and less dense than normal lymphocytes in the FCM analysis. By carefully defining the FCM gating strategy, we were able to establish the approach to analyse both quiescent HSCs (qHSCs) and activated HSCs (aHSCs) in human liver specimens. Importantly, we found that co-expression of CD14 and CD56 within CD45 non-immune cells permits the detection of qHSCs, whereas CD68 and CD40 within CD45 non-immune cells were valuable for assessing aHSCs. Furthermore, we found that aHSCs in PBC upregulated the expression of multiple markers associated with antigen-presentation capacity. Our established approach with FCM will be valuable for the direct analysis of qHSCs and aHSCs with FCM in various human liver specimens. Our FCM analysis of aHSCs in PBC suggested their involvement in the local immune responses.