Fig. 1

Characterization of E-cadherin⁺ fetal liver cells as bipotential progenitor cells. a The experimental schema for p53^−/− hepatoblast isolation, creation of hepatic inflammatory environment, and trans-splenic intrahepatic delivery of hepatoblasts for either liver repopulation or tumor formation. b Expression of hepatic progenitor cell-associated surface markers examined by flow cytometry after 5-day culture with immunohistochemistry confirmation. Histograms represent expression of E-cadherin (upper), CD133 (middle), and EpCAM (lower) relative to isotype controls. Cells cultured on Matrigel-coated were fixed for immunocytochemistry by a two-step indirect protocol for confocal microscopy. Representative images showing expression patterns of E-cadherin, CD133 and EpCAM are shown in the right panel. c Reverse-transcription PCR expression of hepatocytic (AAT1, AFP, ALB, and TDO2), cholangiocytic (G6PC, KRT7, and KRT19) and progenitor cell markers (DLK1, SOX9, EPCAM, MYC, and NANOG) by purified E-cadherin⁺ fetal liver cells after 5 days culture. d Immunofluorescence demonstrating that E-cadherin⁺ fetal liver cells express ALB/CK19 as well as AFP/EpCAM. Upper representative view shows two colonies derived from single E-cadherin⁺ fetal liver cells co-cultured with STO feeders. Lower representative view shows a colony derived from a single E-cadherin⁺ fetal liver cell cultured on Matrigel. f Single E-cadherin⁺ fetal liver cell-derived clones contained exclusively ALB-positive hepatocytes and CK19-positive cholangiocytes after a 7-day spontaneous differentiation after co-culture with STO feeders in basal media. Left image shows a colony manifesting mostly cholangiocellular differentiation. The right image showed bi-directional differentiation. f In vivo repopulation and differentiation of E-cadherin-positive fetal liver cells. The sections of the recipient liver were subjected to observation of GFP-immunoreactive cells forming bile duct structures (upper) and liver parenchyma (lower). Mice received splenic injection of HBSS as controls