Isolation and Characterization of Mouse and Human LSECs

Mouse LSECs were isolated by previously described two-step collagenase perfusion technique with modifications^{15 (link)}. In brief, the liver was perfused with Liver Perfusion Medium (Invitrogen), and dissociated by Liver Digest Medium (Invitrogen). The NPCs were fractionated with Percoll® (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) gradient centrifugation with 75% stock Percoll solution and 35% stock Percoll solution. LSEC faction was isolated by mouse LSEC binding magnetic beads (Miltenyi, Auburn, CA, USA) and Dynabeads® Magnetic Beads conjugated with anti-mouse CD31 antibody (MEC13.3, BD Biosciences). Expression of Id1, CXCR7, HGF, and Wnt2 mRNA were determined. Primary human LSECs were procured from ScienCell Research Laboratories (Catalog #5000, Carlsbad, CA, USA). Expression of factor VIII was validated by immunostaining. Akt-LSECs were derived from isolated LSECs that were transfected with the pCCL. PGK lentiviral vector with mouse constitutively active Akt1 (myristoylated Akt: myrAkt)^{63 (link)}. After starving in serum-free medium, 500,000 LSECs were seeded and stimulated with 10 ng/ml SDF-1. LSECs were also treated with 30 μM Wortmannin (Sigma-Aldrich).