Hepatic maturation of human fetal hepatocytes in four-compartment three-dimensional perfusion culture.

Bio-artificial liver support systems have been utilized as bridging devices to support acute and chronic liver injury. However, prolonged function of adult hepatocytes has not been achieved due to compromised proliferation and long-term survival of adult cells in vitro. As an alternative cell source, we investigated the potential of human fetal hepatocytes (hFH) in a four-compartment hollow fiber-based three-dimensional (3D) perfusion culture system. hFH were isolated from 17- to 19-gestational-week livers and cultured in the 3D perfusion bioreactors for 14 days. Metabolism activity, hepatocyte-specific gene expression, protein expression, and hepatic function were investigated. Increased glucose consumption and lactate production indicated cell proliferation in the bioreactor. The ratio of cytochrome P450 3A4 to 3A7 gene expression and the increase of the number of asialoglycoprotein receptor-positive cells indicated cell differentiation into mature hepatocytes. Histological and immunohistochemical analysis revealed reorganization of fetal liver cells. Hepatic function was further examined for ammonia metabolism and for albumin production using colorimetric assays and enzyme-linked immunosorbent assay, respectively. In contrast to conventional 2D culture, the 3D perfusion culture system induced functional maturation to hFH; these cells may be useful as an alternative cell source for extracorporeal liver support.

Quantitative comparison of stem cell marker-positive cells in fetal and term human amnion.

Scattered in the amniotic epithelium of the human term placenta are pluripotent stem cell marker-positive cells. Unlike other parts of the placenta, amniotic epithelial (AE) cells are derived from pluripotent epiblasts. It is hypothesized that most epiblast-derived fetal AE cells are positive for stem cell markers at the beginning of pregnancy and that the stem cell marker-positive cells scattered through the term amnion are remaining, epiblast-like stem cells. To test this hypothesis, human fetal amnia from early-stage pregnancies were evaluated for expression of the stem cell-specific cell surface markers TRA 1-60 and TRA 1-81 and of the pluripotent stem cell marker genes Oct4, Nanog, and Sox2. Whole-mount immunohistochemical analysis demonstrated that a greater proportion of AE cells in the 17-19 week human fetal amnion are positive for both TRA 1-60 and TRA 1-81 than in the term amnion. Quantitative real-time RT-PCR analysis confirmed that the fetal AE cells exhibit greater stem cell marker gene expression than those in term placentae. Expression of both Nanog and Sox2 mRNAs were significantly higher in the fetal amnion, while Oct4 mRNA expression was not significantly changed. These differences in abundance of stem cell marker-positive cells and stem cell marker gene expression together indicate that some stem cell marker-positive cells are conserved over the course of pregnancy. The results suggest that stem cell marker-positive AE cells in the term amnion are retained from epiblast-derived fetal AE cells.