Isolation and Characterization of Mouse and Human Liver Sinusoidal Endothelial Cells

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 messenger RNA was determined. Primary human LSECs were procured from ScienCell Research Laboratories (catalog no. 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⁻¹ SDF-1. LSECs were also treated with 30 μm Wortmannin (Sigma-Aldrich).