Human placental cell suspensions prepared by trypsin digestion were analyzed with several monoclonal antibodies on a multiparameter fluorescence-activated cell sorter (FACS). Five distinct cell populations were isolated on the basis of size and quantitative differences in the coordinate expression of cell surface antigens detected by monoclonal antibodies against an HLA-A,B,C monomorphic determinant (MB40.5) and against human trophoblasts (anti-Trop-2). By FACS analysis and after sorting we clearly identified the major cell population as cytotrophoblasts based on several independent criteria, including presence of trophoblast-specific surface antigens, Trop-1, and Trop-2; absence of all HLA class I, class II, and beta 2-microglobulin (beta 2m) antigens; absence of the pan-leucocyte and monocyte antigens, HLe1 and LeuM1, respectively; presence of Y-chromatin in a male placenta; presence of placental and not liver alkaline phosphatase; and a large, mononuclear morphology. These procedures provide a reproducible method for obtaining highly purified human cytotrophoblast populations for further studies. We measured by molecular hybridization (RNA or Northern blots) the HLA-A,B,C and beta 2m mRNA in total RNA extracted from sorted cytotrophoblasts. We find that normal human cytotrophoblasts have extremely small amounts of HLA-A,B,C mRNA: approximately 300 times less than that in the lymphoid cell line LCL-721 or normal lymphocytes. In contrast, they have approximately 11% the level of beta 2m mRNA present in LCL-721 cells. Thus, HLA-A,B,C antigen expression on human cytotrophoblasts is limited by the level of HLA heavy chain mRNA.

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