Characterization and enrichment of fetal rat hepatoblasts by immunoadsorption (“panning”) and fluorescence-activated cell sorting

Authors

  • Samuel H. Sigal,

    1. Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
    2. Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
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  • Shlomo Brill,

    1. Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
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  • Lola M. Reid Ph.D.,

    Corresponding author
    1. Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
    2. Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York 10461
    3. Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
    4. Chanin Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461
    • 601 Chanin Cancer Center, 1300 Morris Park Avenue, Bronx, NY 10461
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  • Isabel Zvibel,

    1. Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
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  • Sanjeev Gupta,

    1. Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
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  • Douglas Hixson,

    1. Department of Medical Oncology, Brown University-Rhode Island Hospital, Providence, Rhode Island 02902
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  • Ronald Faris,

    1. Department of Medical Oncology, Brown University-Rhode Island Hospital, Providence, Rhode Island 02902
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  • Patricia A. Holst

    1. Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461
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Abstract

We developed methods for enriching fetal hepatoblasts by combining panning and multiparametric fluorescence-activated cell sorting. In unpurified, dissociated fetal liver cell suspensions of embryonic age day 15, 3.2% ± 1.3% and 2.5% ± 0.7% cells expressed albumin and α-fetoprotein, respectively. The remainder exhibited a hemopoietic, endothelial or stromal cell phenotype. Cells were panned first with an antibody to red blood cells to remove erythroid cells and then with monoclonal antibodies OX-43/OX-44 to remove hemopoietic and endothelial cells. This procedure eliminated 84% of fetal hepatic cells, with enrichment of the remainder for albumin or α-fetoprotein expression (up to sixfold increase). Flow cytometric analysis of unlabeled cells revealed two populations, which differed in granularity and autofluorescence. After panning, fluorescence-activated cell sorting for agranular cells yielded OX-43/44-positive cells that were essentially all hemopoietic precursor cells or OX-43/44-negative cells that were mostly hemopoietic precursor cells, along with 3.0% ± 0.7% α-fetoprotein-positive cells. In contrast, sorting for granular, OX-43/44-negative cells enriched for predominantly α-fetoprotein-positive, parenchymal precursor cells (75.1% ± 4.7%). Multiparametric flow cytometric analysis of the expression of an oval cell antigen, OC.3, which is a bile duct and putative liver stem cell marker, showed that most OC.3-positive cells coexpressed OX-43/OX-44 and morphologically were hemopoietic precursor cells. However, approximately 30% of the OX-43/44-negative, granular cells expressed OC.3. Although the physiological significance of OC.3-positive hepatoblasts remains to be determined, the ability to isolate distinct liver cell populations by means of fluorescence-activated cell sorting should facilitate further studies. In addition, because panning alone produced significantly enriched populations of fetal hepatoblasts, applications not requiring further cell purification could be performed with this simple technique. (HEPATOLOGY 1994;19:999–1006.)

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