Characterization and enrichment of fetal rat hepatoblasts by immunoadsorption ("panning") and fluorescence-activated cell sorting.
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 alpha-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 alpha-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% alpha-fetoprotein-positive cells. In contrast, sorting for granular, OX-43/44-negative cells enriched for predominantly alpha-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.(ABSTRACT TRUNCATED AT 250 WORDS)
Sigal
S H
SH
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461.
Brill
S
S
Reid
L M
LM
Zvibel
I
I
Gupta
S
S
Hixson
D
D
Faris
R
R
Holst
P A
PA
D000418:Albumins / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D000954:Antigens, Surface / Q000032:analysis; D015152:Blotting, Northern; D002448:Cell Adhesion; D002454:Cell Differentiation; D002469:Cell Separation; D048429:Cell Size; D002478:Cells, Cultured; D004727:Endothelium / Q000166:cytology; D015672:Erythroid Precursor Cells / Q000276:immunology; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000276:immunology; D007163:Immunosorbent Techniques; D008099:Liver / Q000166:cytology / Q000196:embryology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D011916:Rats, Inbred F344; D013234:Stem Cells / Q000166:cytology* / Q000276:immunology; D017154:Stromal Cells / Q000166:cytology; D000509:alpha-Fetoproteins / Q000096:biosynthesis / Q000235:genetics
D000418:Albumins; D000954:Antigens, Surface; D012333:RNA, Messenger; D000509:alpha-Fetoproteins