Modulation of hepatitis B virus infection by epidermal growth factor secreted from liver sinusoidal endothelial cells

Authors:
Shin-Wei C hen, Misao Himeno, Yuta Koui, Masaya Sugiyama, Hironori Nishitsuji, Masashi Mizokami, Kunitada Shimotohno, Atsushi Miyajima & Taketomo Kido
In:
Source: Scientific Reports
Publication Date: (2020)
Issue: 10: 14349
Research Area:
Gastroenterology
Stem Cells
Cells used in publication:
Hep G2
Species: human
Tissue Origin: liver
Hepatocyte, mouse
Species: mouse
Tissue Origin: liver
Culture Media:
Experiment

Co-culture system. Hepatocytes derived from iPSCs were in the lower chamber of trans-well. To induce hepatic maturation, cells were incubated in Hepatocyte Basal Medium (LONZA) supplemented with HCM SingleQuots (excluding EGF) and Oncostatin M (20 ng/ml) (PeproTech). NPCs were suspended in Endothelial Cell Growth Basal Medium plus (LONZA) supplemented with VEGF (50 ng/ml) and Y27632 (10 µM) and seeded into the upper chamber of trans-well. Co-culture started at HGF stage. After 10 days of co-culture, cells were infected with HBV for 16 h. HepG2-NTCP cells were suspended in HepG2-NTCP maintenance medium and seeded on a collagen I-coated plate at the density of 25,000 cells/cm2. iPSC-derived NPCs were suspended in maintenance medium and seeded into the upper chamber of trans-well. Co-culture started at day 1, and 6 days of co-culture, cells were infected with HBV for 16 h.

Abstract

Hepatocytes derived from human iPSCs are useful to study hepatitis B virus (HBV) infection, however infection efficiency is rather poor. In order to improve the efficiency of HBV infection to iPSC-derived hepatocytes, we set a co-culture of hepatocytes with liver non-parenchymal cells and found that live sinusoidal endothelial cells (LSECs) enhanced HBV infection by secreting epidermal growth factor (EGF). While EGF receptor (EGFR) is known as a co-receptor for HBV, we found that EGF enhanced HBV infection at a low dose of EGF, whereas EGF at a high dose suppressed HBV infection. EGFR is internalized by clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE ) pathways depending on the dose of EGF. At a high dose of EGF, the endocytosed EGFR via CIE is degraded in the lysosome. This study is the first to provide evidence that HBV is endocytosed via CME and CIE pathways at a low and high dose of EGF, respectively. In conclusion, we developed an in vitro system of HBV infection using iPSC- derived liver cells, and show that EGF secreted from LSECs modulates HBV infection in a dose dependent manner.

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