Transcriptome analysis of primary human hepatocytes-derived organoids [RNA-seq]

In this study, we established human hepatocyte organoids (HHOs) that were expanded from primary human hepatocytes (PHH) in a defined medium. Briefly, the cryopreserved primary human hepatocytes (purchased) were thawn in advanced DMEM/F12 (Thermo) or Cryopreserved hepatocyte Recovery Medium (Gibco). Then, the cells were embedded in Matrigel (Corning) and cultured with the expansion medium (EM). For the eHHOs, expansion medium (EM) was prepared with a basal medium (Advanced DMEM/F12 was supplemented with penicillin/streptomycin, 10 mM HEPES, 2 mM GlutaMAX, 1 ×(Thermo Fisher Scientific), 10 nM gastrin I (Sigma), and 1 mM N-acetylcysteine (Wako, Japan) ) containing the following niche factors: 50 ng/ml mouse recombinant EGF (Thermo Fisher Scientific), 25 ng/mL human recombinant HGF (Peprotech), 100 ng/mL human recombinant FGF-10 (Peprotech), 10 uM Forskolin (Cayman Chemical), 25 ng/ml mouse recombinant noggin (Peprotech), 1 mg/ml human recombinant R-spondin1 (R; R&D), 20% Afamin-Wnt-3A serum-free conditioned medium (W; Mihara et al., 2016), 5 uM A83-01 (Tocris), and 20ng/ml human Oncostatin M (OSM; Peprotech). For dHHOs, eHHOs were cultured for 10-14 days in differentiation medium (DM), which was EM without OSM and WR, and containing a hormone cocktail (10ng/ml Growth Hormone, 10ng/ml Prolactin, and 100 ng/ml Cortisol) and 10uM DAPT (differentiation medium: DM).