115 CYTOLOGICAL ANALYSIS OF HEPATIC GENE EXPRESSION AND IMMUNOLOGICAL RESPONSE OF MHC ANTIGENS IN MOUSE AMNIOTIC EPITHELIAL CELLS

Abstract

Human amniotic epithelial cells (hAECs) have been reported to have unique properties. They express almost no class I and class II MHC antigens and lack response to interferon-γ (IFN-γ) which mediates the expression of those MHC molecules. Moreover, hAECs express some genes characteristic of hepatic cells. Therefore, hAECs seem to have multipotency and are expected to substitute for hepatic tissues in part. We aimed to develop the experimental model for investigating AECs in mice (mAECs). In this study, we examined the induction of MHC molecules by IFN-γ and the hepatic gene expression in mAECs. Murine amniotic membranes were collected from C57BL/6J females at 17.5 days of gestation. They were digested by 0.03% hyaluronidase followed by 0.2% collagenase treatment. Dissociated mAECs were cultured on dishes in DMEM supplemented with 10% FBS at 37°C under 5%CO₂ in air. Embryonic fibroblasts (EFs) collected from C57BL/6J fetuses at 13.5 dpc were cultured in the same condition as mAECs. In Experiment I, the effect of IFN-γ on induction of MHC molecules in mAECs was examined. mAECs and EFs cultured in the presence or absence of IFN-γ at 1 × 10³ U mL⁻¹ for 72 h were recovered and incubated with FITC-conjugated antibodies against mouse H-2 MHC class I or I-A/I-E MHC class II antigens. The cells were analyzed by flow cytometry. In Experiment II, the expression of the genes in mAECs was examined by RT-PCR. mRNA was purified from adult liver, EFs, fresh mAECs, and mAECs cultured for 5 days. As the genes characteristic for hepatic cells, HNF-3α, HNF-3β, HNF-3γ, HNF-4, transthyretin (TTR), albumin, α-fetoprotein (AFP), glucose-6-phosphatase (G6P), and asialoglycoprotein receptor-1 (Asgr1) were examined. In Experiment I, cell-surface expression of class I and class II MHC antigens in response to IFN-γ was observed weakly in mAECs as compared to EFs, suggesting different property in hAECs which lack the expression of those antigens. In Experiment II, RT-PCR analysis showed that all of the genes except G6P were expressed in fresh mAECs. However, the expression of transcription factors such as HNF-3α, HNF-3β, HNF-4, and TTR, serum proteins such as albumin and AFP, and Asgr1 decreased after in vitro culture, contrary to the case of hAECs in which, for example, albumin appeared after cultivation. In conclusion, it was evident that mAECs have quite different properties, both in the inductivity of MHC molecules and the expression of hepatic genes, from hAECs.

This work was supported by Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence of the JST, and by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT.