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Isolation of human progenitor liver epithelial cells with extensive replication capacity and differentiation into mature hepatocytes

¹ Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
² Marion Bessin Liver Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
³ Cancer Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
⁴ General Clinical Research Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA

View larger version (152K): [in a new window]   Fig. 1. Selected properties of progenitor liver cells in culture. (A) shows G-6-P, glycogen, DPPIV and GGT expression immediately after isolation of hepatoblasts. The arrow in the lower left panel indicates a binucleated hepatoblast. (B) Electron microscopy of cultured cells in the third passage (P3) showing epithelial morphology with characteristic nuclei and prominent nucleoli. The inset shows microvilli (arrows) and bile canalicular-type structure in two adjacent cells, which was in agreement with the epithelial morphology of the cells. Bar, 2 µm. (C) Primary culture of isolated cells with expression of various liver markers. These cells had been in culture for 3 weeks with depletion of non-adherent cells from the culture. (D) The fifth passage culture, showing heaped up cells expressing liver genes, which is identical to primary cultures. (E and F) show colocalization of glycogen (purple color) and CK-19 (brown color, arrows) in P1 progenitor cells arranged as monolayers (E) or cell heaps (F). The cells were isolated from livers #21198, #32593 and 210991.

View larger version (26K): [in a new window]   Fig. 2. Gene expression in cultured progenitor cells. (A) Western blots showing liver gene expression in primary cells (Pr) and eighth passage cells (P8), including cell lysates or culture medium. -1 antitrypsin, CK-8 and CK-19 were not expressed in eighth passage cells. Arrowheads indicate monomeric and dimeric forms of orosomucoid. (B) PAI-1 expression with SDS-PAGE showing metabolic labeling with 35S methionine and cysteine (left panel). Compared with HepG2 cancer cells, eight passage progenitor cells showed increased 43 kDa protein expression. Peptide mass spectroscopy suggested that this band was PAI-1, which was verified by western blotting (C), which showed PAI-1 expression in primary and subpassaged progenitor cells but not in HepG2 cells (right panel).

View larger version (48K): [in a new window]   Fig. 3. Proliferation in cultured progenitor cells. (A) Cell colonies formed by third passage progenitor cells under limiting dilution conditions. The top left panel shows phase microscopy. Other panels show glycogen, G-6-P and GGT expression. (B) Growth factor responsiveness of progenitor cells. DNA synthesis rates following stimulation with HGF, TGF and EGF increased in rat hepatocytes but progenitor cells responded to TGF and EGF only.

View larger version (96K): [in a new window]   Fig. 4. In vivo fate of progenitor liver cells. Eighth passage cells are shown 3 weeks after transplantation into the peritoneal cavity. (A) Confluent mass of transplanted cells adjacent to microcarrier bead (m), with typical hepatocyte morphology (H&E stain). (B) In situ hybridization of contiguous sections with human DNA probe visualized cell nuclei, whereas negative control tissues, where the probe was omitted, showed no signal (C). Transplanted cells contained G-6-P (D), glycogen (E), -1 microglobulin (F) and albumin (G). The arrow in E points toward glycogen granules. (H) shows electron micrography of transplanted cells adhering to a microcarrier bead (m). The cells display epithelial morphology with microvilli on the apical surface (arrow) plus nuclei, nucleolus, abundant mitochondria and bile canaliculi (bc), which are ypical of hepatocytes (original magnification x2,500).

View larger version (159K): [in a new window]   Fig. 5. Progenitor cells engrafted and proliferated in the mouse liver. (A) In situ hybridization showing nuclear localization of the signal in fetal human liver. (B) Negative control tissue showing absence of hybridization when the probe was omitted. (C) shows transplanted cells with dark nuclear staining (arrow) in a portal area (Pa) of recipient mouse liver after 24 hours. (D and F) show integration of transplanted cells in the liver six weeks after transplantation with transplanted cells localized by in situ hybridization (arrows) and histochemistry showing either G-6-P expression (D) or bile canalicular ATPase activity (arrowheads, E). (F) Only occasional transplanted cells were observed as shown six weeks after cell transplantation. (G) shows the increased number of transplanted cells six weeks after repeated CCl4 treatment to induce cell proliferation. Panels C, F and G were counterstained with methyl green.