Sigal S H, Brill S, Reid L M, Zvibel I, Gupta S, Hixson D, Faris R, Holst P A
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461.
Hepatology. 1994 Apr;19(4):999-1006.
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)
我们通过结合淘选法和多参数荧光激活细胞分选技术，开发出了富集胎儿肝母细胞的方法。在胚胎第15天未纯化的解离胎儿肝细胞悬液中，分别有3.2%±1.3%和2.5%±0.7%的细胞表达白蛋白和甲胎蛋白。其余细胞呈现造血、内皮或基质细胞表型。细胞首先用抗红细胞抗体进行淘选以去除红细胞，然后用单克隆抗体OX - 43/OX - 44去除造血和内皮细胞。此过程消除了84%的胎儿肝细胞，使剩余细胞中白蛋白或甲胎蛋白表达得以富集（增加了六倍）。对未标记细胞的流式细胞术分析显示有两个群体，其在颗粒度和自发荧光方面存在差异。淘选后，对无颗粒细胞进行荧光激活细胞分选，得到的OX - 43/44阳性细胞基本上都是造血前体细胞，或者OX - 43/44阴性细胞，其中大多也是造血前体细胞，还有3.0%±0.7%的甲胎蛋白阳性细胞。相比之下，对有颗粒的OX - 43/44阴性细胞进行分选，主要富集了甲胎蛋白阳性的实质前体细胞（75.1%±4.7%）。对一种卵圆细胞抗原OC.3（一种胆管和假定的肝干细胞标志物）表达的多参数流式细胞术分析表明，大多数OC.3阳性细胞共表达OX - 43/OX - 44，形态上是造血前体细胞。然而，约30%的OX - 43/44阴性有颗粒细胞表达OC.3。虽然OC.3阳性肝母细胞的生理意义尚待确定，但通过荧光激活细胞分选分离不同肝细胞群体的能力应有助于进一步研究。（摘要截选至250字）
