Self-renewal of hepatoblasts under chemically defined conditions by reiterative growth factor and chemical screening

Throught an reiterative growth factor and chemical screening, we defined a small molecule and growth factor cocktail, including EGF, glycogen synthase kinase 3 inhibitor (CHIR99021), transforming growth factor β receptor inhibitor (e.g. E-616452), lysophosphatidic acid and sphingosine 1-phosphate, that can sustain long-term self-renewal of murine hepatoblasts under chemically defined conditions. The expandable hepatoblasts (eHBs) by this small molecule and growth factor cocktail expressed a set of genes typical of liver progenitor cells and liver development, and retained the ability to respond to liver developmental cues and produce functional hepatocytes and form bile duct-like structures. Moreover, both early- and late-passage eHBs demonstrated a similar transcriptome profile. Microarray analysis also confirmed that the gene expression of cultured cells resembled hepatoblasts. Expandable hepatoblasts (eHBs) were routinely cultured in basal medium (DMEM/F12 medium supplemented with insulin-transferrin-sodium selenite, 5 mM nicotinamide, 30 μg/ml 2-phospho-L-ascorbic acid and 50 μg/ml bovine serum albumin human recombinant albumin and 1% penicillin/streptomycin including 10 ng/ml EGF, 3 μM CHIR99021, 2 μM E-616452, 5 μM LPA and 0.5 μM S1P, on Matrigel or laminin-coated surface. The culture was split 1:6 using Accutase. Total RNA was prepared from passage 5 and passage 20 of eHBs (passage 5 and passage 20 of eHBs were derived from two independent experiments) using the RNeasy Plus Mini Kit.