|Budget Amount *help
¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1992 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Although the fetal liver has been considered to be the main hemopoietic organ in the embryonal period, the role of hepatocytes in hemopoiesis remains obscure. We have established an epithelial cell line from the murine fetal liver which can support hemopoiesis in vitro. The cells were identified as epithelial cells by the presence of desmosomes and tight junctions. Other morphological features also helped in confirming the hepatic origin of the cell line (designated as FHC). The cells in the primary culture were positively stained with both antibodies against murine a-fetoprotein and against murine albumin, indicating the hepatocytic nature of the cell line. Cloned FHC (FHC-4D2) cells were demonstrated to have the ability to maintain hemopoietic progenitors in the fetal liver and adult bone marrow in the coculture. At least a part of hemopoiesis supporting function was found to be mediated by the soluble factors the cell produced. Column chromatography, neutralization test with antibod
ies, and the proliferation assay demonstrated that the hemopoietic activities in the 4D2 supernatant were attributed to GM-CSF and M-CSF. Transcripts of GM-CSF and M-CSF were detected in Northern blot analysis, whereas no messages for IL-3, IL-6, G-CSF or erythropoietin were identified. Hemopoietic progenitor cells were well maintained for at least 12 weeks without any growth factors, suggesting that, in addition to CSFs that 4D2 produced, 4D2 could exert the hemopoiesis-supporting function through direct contact with hemopoietic cells. The coculture condition It was also found that when fetal thymocytes were co-cultured with 4D2 clone in the presence of rIL-2, they proliferated for at least two weeks. Proliferating cells in the co-culture of fetal thymocytes contained 4-12% TCR-bearing cells, among which TCR gammadelta^+ cells were predominant: the majority of TCR gammadelta^+ cells were found to use Vgamma5. About half of CD3^+ cells expressed Lyt-2, but not Lyt-3 or L3T4, indicating that they were CD8alphaalpha^+ cells. Additionally, the proliferating cells in the co-culture displayed both NK-like and TCR-mediated cytolytic activities. Moreover, 4D2 cells have differentiated and maintained immature B cells from the bone marrow or fetal liver.
Taken together, these results suggest that 4D2 clone could promote hemopoiesis and lymphopoiesis for both T and B lymphocytes. Since FHC-4D2 is the first fetal hepatocyte cell line capable of supporting hemopoiesis, this study could directly demonstrate for the first time that hepatocytes play an active role in the fetal hemopoiesis as well as maintaining and differentiating both T and B lymphocytes in the fetal and the newborn livers. This co-culture system might be very useful as an in vitro tool to explore the role of fetal hepatocytes in the fetal liver hemopoiesis.