Neonatal LSCs of passage 9 (one passage corresponds to approximately three cell population doublings for these cells according to the manufacturer’s protocol) from the 1-year-old donor were purchased from Kerafast Inc. The commercial LSCs were cultured in polarization media [Dulbecco’s modified Eagle’s medium, 10% dialyzed fetal bovine serum (Invitrogen), 1.5 mM xanthosine (Sigma), 1× penicillin/streptomycin, epidermal growth factor human (20 ng/ml; Invitrogen), transforming growth factor–β human recombinant (0.5 ng/ml Sigma)] according to the manufacturer’s protocol (Kerafast Inc.) (3941). These cells served as controls to characterize de novo isolated and polarized LSCs.

Additional LSC cultures were isolated and polarized and characterized from the bulk commercial hepatocyte suspensions (Lonza Walkersville Inc.) from young donors using previously described protocols with specific modifications (14, 15) combined with the aforementioned Kerafast protocol for neonatal LSCs. Briefly, bulk suspension hepatocytes (0.5 × 106 to 1 × 106 of cells) were transferred to polarization media as described for the neonatal LSCs and cultured on cell-adhesive 12-well plates for 5 to 7 days. Then, all nonattached hepatocytes were removed, and fresh media were added to the small remaining population of attached progenitor cells. After 1 to 1.5 weeks of culture and media changes, attached cells symmetrically divided, growing to mixed clonal populations of polarized adult LSCs. These cultures were frozen at early passage (p = 3 to 5) until further use. Only LSCs from donors of younger age (≤22 years) could be isolated in this way.

Phenotypes of the polarized cells were analyzed for the presence of specific surface stem cell and epithelial progenitor cell epitopes, e.g. EpCAM (epithelial cell adhesion molecule), Lgr5, CD90, CD29, CD105, and CD73, upon staining with antibodies by means of multicolor flow cytometry analysis (LSRII, Becton Dickinson) as recommended previously (14, 15, 42, 43). Characteristic FACS profiles and specific phenotypes for commercial LSCs (control) and two manually isolated and polarized LSC lineages are shown in fig. S1B.

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