Hepatic oval cells have the side population phenotype defined by expression of ATP-binding cassette transporter ABCG2/BCRP1

Abstract

Organ-specific stem cells can be identified by the side population (SP) phenotype, which is defined by the property to effectively exclude the Hoechst 33342 dye. The ATP-binding cassette transporter ABCG2/BCRP1 mediates the SP phenotype. Because hepatic oval cells possess several characteristics of stem cells, we examined whether they have the SP phenotype using the 2-acetylaminofluorene/partial hepatectomy (PH) model. Fluorescence-activated cell sorting analysis showed that a population of non-parenchymal cells containing oval cells, prepared on day 7 after PH, carried a significant number of SP cells, whereas that of non-parenchymal cells without oval cells, prepared on day 0 after PH, did not. Northern blot analysis using total liver RNA obtained on various days after PH showed that the expression of ABCG2/BCRP1 mRNA increased after PH, reaching the highest level on day 7, and then gradually decreased. This pattern of changes in the ABCG2/BCRP1 mRNA level was well correlated to that in the number of oval cells. Furthermore, in situ hybridization revealed that oval cells were the sites of expression of ABCG2/BCRP1 mRNA. These results indicate that oval cells have the SP phenotype defined by expression of ABCG2/BCRP1, suggesting that oval cells may represent stem cells in the liver.

Figure 1.

SP profile of non-parenchymal cells containing oval cells. Non-parenchymal cells were isolated on day 7 after PH in the AAF/PH model, and analyzed for Hoechst 33342 efflux by FACS vantage. A: A clear SP is visible after Hoechst 33342 staining. B: Dye efflux from SP cells is inhibited in the presence of 100 μmol/L verapamil. SP-gated cells are 2.0% of the total number of cells analyzed in A, and 0.05% in B.

Figure 2.

Rat ABCG2/BCRP1 cDNA. A: Schematic representation. An open reading frame of ABCG2/BCRP1 cDNA is indicated by box. Six overlapping partial clones are shown below with primers. B: Nucleotide sequence (GenBank accession no. AB094089). The DNA sequence is numbered from the initiation codon. The predicted amino acid sequence is displayed under the DNA sequence.

Figure 3.

Expression of ABCG2/BCRP1 mRNA. A: Expression of ABCG2/BCRP1 mRNA in various tissues of adult rats. A membrane of rat polyA⁺ RNA (2 μg) Northern blot-12 major tissues was used for Northern blot analysis. GAPDH mRNA was analyzed as an internal standard. B: Time course analysis of CK-19-positive oval cells in livers following PH in the AAF/PH model. Rats were sacrificed on the indicated days after PH, and liver sections were immunostained with an anti-CK-19 antibody. The number of CK-19-positive oval cells was counted in ten periportal fields, and its value was expressed as the number per periportal field (0.25 mm × 0.25 mm). Each point represents the mean of three rats. Bars are standard errors (SE); the SE was sometimes too small to be shown by bars. C: Time course analysis of ABCG2/BCRP1 mRNA in livers following PH in the AAF/PH model. Total liver RNA (20 μg) from rats sacrificed on the indicated days after PH was analyzed by Northern blotting using ABCG2/BCRP1 cDNA as a probe. RNA from rats that did not undergo AAF/PH treatment was used as the control (C). Parenchymal cell fraction (P) and non-parenchymal cell fraction (NP) were isolated from livers on day 7 after PH, and total RNA (20 μg) was analyzed by Northern blotting. 18S and 28S were used as an internal standard. D: A section of the liver obtained on day 7 after PH stained with hematoxylin and eosin. The representative clusters of oval cells are shown by arrows. E: in situ hybridization analysis of ABCG2/BCRP1 mRNA using an adjacent section of D. Inset in E is high magnification, and shows oval cells expressing ABCG2/BCRP1 mRNA. Magnification, ×80 (D and E) and ×750 (inset of E).