title: Proliferation of hepatocyte progenitor cells isolated from adult human livers in serum-free medium.
abstract: Rat small hepatocytes (SHs) are committed progenitor cells that can differentiate into mature hepatocytes and can selectively proliferate in serum-free medium when they are cultured on hyaluronic acid (HA)-coated dishes. In this study we examined the separation of human SHs from adult human livers. We obtained liver tissues from the resected liver of 16 patients who underwent hepatic resections. Extracted liver specimens were clearly separate from the tumor regions with sufficient margins. Hepatic cells were isolated using the modified method of two-step collagenase perfusion. A low-speed centrifugation was performed and cells in the supernatant were finally cultured on HA-coated dishes in serum-free DMEM/F12 medium including nicotinamide, EGF, and HGF. Small-sized hepatocytes selectively proliferated to form colonies and many colonies continued growing for more than 3 weeks. The average number of cells in a colony was 38.6 +/- 18.0, 79.0 +/- 54.0, and 101.5 +/- 115.7 at day 7, 14, and 21, respectively. About 0.04% of plated cells could form an SH colony. Immunocytochemistry showed that the cells forming a colony were positive for albumin, transferrin, keratin 8, and CD44. The results of RT-PCR showed that colony-forming cells expressed albumin, transferrin, alpha1-antitrypsin, fibrinogen, glutamine synthetase, many cytochrome P450s, and liver-enriched transcription factors (HNF3alpha, HNF4alpha, C/EBPalpha, and C/EBPbeta). Furthermore, the cells expressed not only the genes of hepatic differentiated functions but also those of both hepatic stem cell marker (Thy1.1, EpCAM, AFP) and SH marker (CD44, D6.1A, BRI3). Albumin secretion into culture medium was also observed. Our results demonstrate the existence of hepatocyte progenitor cells in human adult livers, and the cells can grow in a serum-free medium on HA-coated dishes. Human SHs may be a useful source for cell transplantation as well as pharmaceutical and toxicological investigations.
authors.affiliation: Department of Pathophysiology, Cancer Research Institute, Sapporo Medical University School of Medicine, Sapporo 060-8556, Japan.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Sasaki
authors.forename: Junko
authors.initials: J
authors.lastname: Kon
authors.forename: Toru
authors.initials: T
authors.lastname: Mizuguchi
authors.forename: Qijie
authors.initials: Q
authors.lastname: Chen
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Ooe
authors.forename: Hideki
authors.initials: H
authors.lastname: Oshima
authors.forename: Koichi
authors.initials: K
authors.lastname: Hirata
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Mitaka
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000418:Albumins / Q000378:metabolism; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002478:Cells, Cultured; D020099:Coated Materials, Biocompatible / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005260:Female; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D006820:Hyaluronic Acid / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D013048:Specimen Handling / Q000379:methods; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*
chemical_list: D000418:Albumins; D020099:Coated Materials, Biocompatible; D016895:Culture Media, Serum-Free; D006820:Hyaluronic Acid; D003577:Cytochrome P-450 Enzyme System