Characterization of human HSCs in NASH

Background: Obesity and insulin resistance are common causes non-Alcoholic Steatohepatitis (NASH) characterized by steatosis, ballooning degeneration of hepatocytes, lobular inflammation, activation of hepatic myofibroblasts, and fibrosis development). Human Hepatic Stellate Cells (hHSCs) are the major source of Collagen Type I producing myofibroblasts in fibrotic livers of patients with NASH. The mechanism of hHSC activation in NASH patients is not fully understood, mainly due to the lack of studies utilizing primary human HSCs (hHSCs). Our laboratory has a unique opportunity to obtain livers that are declined for transplantation (collaboration with OPO LifeSharing). AIM: To identify new “marker genes” that distinguish NASH-activated from NAFL(steatoosis)- and normal hHSCs. Methods: Obtained livers were graded by a pathologist using histopathology and biochemical parameters. Primary hHSCs were isolated from livers of donors with no history of liver disease (normal n=8), NAFLD (n=9), and NASH (n=10) using pronase/collagenase and gradient centrifugation, cultured, and analyzed using RNA-Seq. Expression of selected genes was confirmed by qRT-PCR using TGFb1-stimulated hHSCs. Results: Comparative transcriptome analysis revealed that NASH-activated hHSCs and NAFL hHSCs share expression of 1939 genes with normal hHSCs. Out of 1939 genes, only 388 genes were commonly shared between NASH and NAFL hHSCs, suggesting that normal and NAFL hHSCs exhibit high similarity. In turn, NASH-activated hHSCs upregulated 661 genes (APOE, CD14, ROR2, ICAM2, HTRA1) and downregulated 1278 genes (ADAM10, PIK3C2A, LMO3, FADS1, RASSF8, compared to normal hHSCs). Gene Ontology identified pathways associated with human liver diseases (Liver Cirrhosis, Liver Fibrosis, Fatty Liver), inflammation, and fibrogenic activation in NASH hHSCs. Within Liver Fibrosis/Cirrhosis pathways, we observed strong upregulation of CD53, TIMP1, UBD, TYROBP, CD53, TIMP1, APOE, FGFR2 genes, while ADAM17, ADAM10, PDGFRa, IL-33, EGF were downregulated. Moreover, we identified genes that distinguish NASH hHSCs from NAFL hHSCs. NASH-activation of hHSCs is associated with increased expression of 129 genes (including ALKBH5, ECM1, CD9) and suppression of 259 genes (such as PPARGC1A, EBF3, CCR7). Conclusion: Our study provides a new insight into the transcriptomics of primary cultured NASH hHSCs. We characterized the unique features that distinguish NASH hHSCs from normal and NAFL hHSCs. These findings may identify potential novel candidate targets for anti-fibrotic therapy, or used as a readout for drug screening using primary cultured NASH hHSCs.