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Proliferation of Hepatocyte Progenitor Cells Isolated From Adult Human Livers in Serum-Free Medium

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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, α1-antitrypsin, fibrinogen, glutamine synthetase, many cytochrome P450s, and liver-enriched transcription factors (HNF3α, HNF4α, C/EBPα, and C/EBPβ). 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.
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Keywords: CD44; Hepatic differentiated functions; Hepatic stem cells; Hyaluronic acid; Small hepatocytes

Document Type: Research Article

Publication date: 2008-10-01

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  • Cell Transplantation publishes original, peer-reviewed research and review articles on the subject of cell transplantation and its application to human diseases. To ensure high-quality contributions from all areas of transplantation, separate section editors and editorial boards have been established. Articles deal with a wide range of topics including physiological, medical, preclinical, tissue engineering, and device-oriented aspects of transplantation of nervous system, endocrine, growth factor-secreting, bone marrow, epithelial, endothelial, and genetically engineered cells, among others. Basic clinical studies and immunological research papers are also featured. To provide complete coverage of this revolutionary field, Cell Transplantation will report on relevant technological advances, and ethical and regulatory considerations of cell transplants. Cell Transplantation is now an Open Access journal starting with volume 18 in 2009, and therefore there will be an inexpensive publication charge, which is dependent on the number of pages, in addition to the charge for color figures. This will allow work to be disseminated to a wider audience and also entitle the corresponding author to a free PDF, as well as prepublication of an unedited version of the manuscript.

    Cell Transplantation is now being published by SAGE. Please visit their website for the most recent issues.

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