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. Author manuscript; available in PMC: 2017 Feb 16.
Published in final edited form as: Hepatology. 2015 Nov 23;63(1):217–232. doi: 10.1002/hep.28274

Fig. 3.

Fig. 3

Isolated αvβ6+ cells express progenitor (oval) cell markers and differentiate into cholangiocytes and functional hepatocytes in vitro. (A) Freshly isolated αvβ6+ liver cells coexpress hepatocyte, cholangiocyte, and progenitor (oval) cell markers. αvβ6+ cells were purified from a nonparenchymal liver cell fraction of Mdr2−/− mice using magnetic cell sorting and analyzed in comparison with the remaining (αvβ6) cell fraction. αvβ6+ cells (closed bars) overexpress hepatic progenitor markers (Trop2, CD133, EpCAM, α-fetoprotein (Afp), Sox9, Fn14), as well as cholangiocyte (CK19, EpCAM) and hepatocyte (albumin and TAT) markers. Data are expressed as mean ± standard error of the mean. *P < 0.05. (B) Primary αvβ6+ cells form cell colonies and differentiate into both cholangiocyte-like and hepatocyte-like cells in vitro. Representative phase-contrast images from day 1 and 14 in vitro. Original magnification ×400. (C) Up-regulation of cholangiocyte-specific and hepatocyte-specific markers in cultured αvβ6+ cells. Total RNA was extracted at days 0, 7, and 14 and analyzed for cholangiocyte (CK19, HNF1β) and hepatocyte (albumin, HNF4α) markers using RT-PCR. Data are expressed as mean ± standard error of the mean, fold to day 0 (from four individual mice). *P < 0.05. (D) Double-immunofluorescence for αvβ6 integrin and CK19 demonstrate emergence of two distinct cell lineages in αvβ6+ cell colony formation assay. All cells were αvβ6-positive (red), and the majority coexpressed the cholangiocyte marker CK19 (green). CK19-negative cells represented a significant proportion of cells within colonies and demonstrate hepatocyte-like morphology with large and often diploid nuclei (arrows). Representative images of a single large colony derived from αvβ6+ cells after 14 days in culture (original magnification ×200). (E) Albumin levels in cell colony supernatants collected at days 7 and 14 (mouse albumin enzyme-linked immunosorbent assay). Abbreviations: αFP, α-fetoprotein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; K19, cytokeratin 19.