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

• Published in: Hepatology (Baltimore, Md.) Md.), 1994, Vol.19 (4), p.999-1006
• Main Authors: Sigal, Samuel H., Brill, Shlomo, Reid, Lola M., Zvibel, Isabel, Gupta, Sanjeev, Hixson, Douglas, Faris, Ronald, Holst, Patricia A.
• Format: Article
• Language: English
• ISSN: 0270-9139; 1527-3350
• DOI: 10.1016/0270-9139(94)90301-8

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.