The stemness of hepatocytes is maintained by high levels of lipopolysaccharide via YAP1 activation

Background The liver possesses a powerful regeneration ability, which is correlated with the stemness of hepatocytes in the portal vein (PV). However, the mechanism underlying the maintenance of hepatocyte stemness has not been elucidated. Here, we hypothesized that high levels of lipopolysaccharide from the portal vein might maintain the stemness of hepatocytes in the PV area. Methods First, we examined the location of hepatic stem cells and the concentration of lipopolysaccharide (LPS) in the portal vein and inferior vena cava. Then, we assessed the effect of LPS on stemness maintenance in mice by using antibiotics to eliminate LPS and knocking out the LPS receptor, TLR4. In vitro, the effect of LPS on the stemness of hepatocytes was investigated by colony and sphere formation assays and assessment of pluripotent and stem cell marker expression. Furthermore, we studied the mechanism by which LPS regulates the stemness of hepatocytes. Finally, we ligated the portal vein branch to further verify the effect of LPS. Results We found that a high level of LPS from the portal vein was correlated with the location of hepatic stem cells in the PV area, and elimination of LPS by antibiotics inhibited the expression of the stemness marker. LPS promoted colony and sphere formation and induced the upregulation of pluripotent and stem cell markers in AML12 cells. Furthermore, in the reprogramming medium, LPS facilitated the dedifferentiation of mature hepatocytes into hepatic progenitor-like cells, which exhibited a bipotent differentiation capacity in vivo and in vitro. Mechanistically, LPS bound TLR4 to regulate stemness of hepatocytes via the activation of YAP1 signaling, and blockade of YAP1 abolished the LPS-induced cell stemness and upregulation of pluripotent markers. Conclusions Our study implies a correlation between LPS/TLR4/YAP1 signaling and cell stemness, and LPS was shown to be involved in stemness maintenance of hepatocytes in the PV area. LPS might be used to induce the dedifferentiation of mature hepatocytes into progenitor-like cells for repair of liver injury.

Hepatic stellate cells contribute to liver regeneration through galectins in hepatic stem cell niche

Background As a critical cellular component in the hepatic stem cell niche, hepatic stellate cells (HSCs) play critical roles in regulating the expansion of hepatic stem cells, liver regeneration, and fibrogenesis. However, the signaling of HSCs, particularly that involved in promoting hepatic stem cell expansion, remains unclear. While the overexpression of galectins has been identified in regenerating liver tissues, their involvement in cell-cell interactions between HSCs and hepatic stem cells remains to be elucidated. Methods To generate a liver regeneration rat model and establish a hepatic oval cell microenvironment as a stem cell niche, 2-acetylaminofluorene treatment plus partial hepatectomy was performed. Immunofluorescence staining was conducted to detect the emergence of hepatic stem cells and their niche. Liver parenchymal cells, non-parenchymal cells, and HSCs were isolated for gene and protein expression analysis by qPCR or western blotting. To evaluate the effect of galectins on the colony-forming efficiency of hepatic stem cells, c-Kit−CD29+CD49f+/lowCD45−Ter-119− cells were cultured with recombinant galectin protein, galectin antibody, galectin-producing HSCs, and galectin-knockdown HSCs. Results Following liver injury, the cytokeratin 19+ ductal cells were robustly induced together with the emergence of OV6+CD44+CD133+EpCAM+ hepatic stem cells. The activated desmin+ HSCs were recruited around the periportal area and markedly enriched in the galectin-positive domain compared to the other non-parenchymal cells. Notably, the HSC fraction isolated from regenerating liver was accompanied by dramatically elevated gene and protein expression of galectins. Hepatic stem cells co-cultured with HSCs significantly enhanced colony-forming efficiency. Conversely, single or double knockdown of galectin-1 and galectin-3 led into a significant function loss, impaired the co-cultured hepatic stem cells to attenuated colony size, inhibited colony frequency, and reduced total cell numbers in colonies. On the other hand, the promotive function of galectins was further confirmed by recombinant galectin protein supplementation and galectins blocking antibodies. Conclusions Our findings, for the first time, demonstrated that galectins from activated HSCs contribute to hepatic stem cell expansion during liver regeneration, suggesting that galectins serve as important stem cell niche components.