Summary
Hepatic stellate cells (HSCs) play a central role in the progression of liver fibrosis by producing extracellular matrices. The development of drugs to suppress liver fibrosis has been hampered by the lack of human quiescent HSCs (qHSCs) and an appropriate
in vitro
model that faithfully recapitulates HSC activation. In the present study, we developed a culture system to generate qHSC-like cells from human-induced pluripotent stem cells (hiPSCs) that can be converted into activated HSCs in culture. To monitor the activation process, a red fluorescent protein (
RFP
) gene was inserted in hiPSCs downstream of the activation marker gene actin alpha 2 smooth muscle (
ACTA2
). Using qHSC-like cells derived from RFP reporter iPSCs, we screened a repurposing chemical library and identified therapeutic candidates that prevent liver fibrosis. Hence, hiPSC-derived qHSC-like cells will be a useful tool to study the mechanism of HSC activation and to identify therapeutic agents.
HBV attachment assay. HBV derived from the culture supernatant of HepAD38 cells. Cells were incubated with the virus at 10,000 GEq/cell in the presence of 4% PEG8000 and 2% DMSO at 4 °C for 3 h. After washing out the free virus, HBV DNA was detected by qPCR. Primers are listed in Supplementary information. HBV internalization assay. Following HBV attachment assay, the cells were washed to remove free virus and incubated at 37 °C for 24 h. The attached virus was removed by 0.25% trypsin/EDTA and HBV DNA was detected by qPCR. Cytokine array analysis. Human Cytokine Antibody Array Membrane (Abcam) was used to detect cytokines according to the manufacturer's protocol. iPSC-derived LSECs were suspended in NPC maintenance medium and seeded at the density of 40,000 cells/cm 2 in the upper chamber of trans-well. Control wells contained NPC maintenance medium only. HepG2-NTCP maintenance medium was added to the lower chamber and changed to fresh medium every day. The samples were prepared from the lower chamber at day 5, and the data were analyzed by ImageJ quantitatively. The pictures were taken in ImageQuant LAS 4000 version1.2. Cross-linking assay. In cross-linking assay, the cells were incubated with the cross-linker BS 3 at 4 °C for 30 min, and the cross-linking reaction was terminated using 20 mM of glycine (final concentration) as previously described 11. Collected and isolated the cell surface lysates using Pierce Cell Surface Protein Isolation Kit (Thermo Fisher Sciences). Samples were then separated on SDS-PAGE, performed western blotting. Antibodies are listed in Supplementary information. Knockdown of EGFR. Sequences of siRNAs used for knockdown EGFR are: si-EGFR: 5′-GGA ACU GGA UAU UCU GAA A TT-3′ and 5′-GAU CUU UCC UUC UUA AAG A TT-3′. Cells were transfected with siRNAs at a final concentration of 30 nM using Lipofectamine RNAiMAX Reagent (Life Technologies). The Mission siRNA Universal Negative control were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cells were used for experiments 48 h post-transfection.
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