Volume 39, Issue 2, Pages 162-170 (August 2003) Purification of adult hepatic progenitor cells using green fluorescent protein (GFP)- transgenic mice and fluorescence-activated cell sorting  Takahisa Fujikawa, Tetsuro Hirose, Hideaki Fujii, Shoshiro Oe, Kentaro Yasuchika, Hisaya Azuma, Yoshio Yamaoka  Journal of Hepatology  Volume 39, Issue 2, Pages 162-170 (August 2003) DOI: 10.1016/S0168-8278(03)00237-X

Fig. 1 Characterization of GFP-positive cells from the adult liver of selected GFP-transgenic mouse strain. Whole dissociated cells were cultured in our standard medium for 7 days. The figure shows findings of the phase contrast (A, C, E) and corresponding fluorescent images (B, D, F): (D) merged image for GFP expression and α-SMA (red) staining; (F) merged image for GFP, acetylated LDL (red) and latex beads (blue). (A, B) Mature hepatocytes strongly expressed GFP. (C, D) Immunocytochemistry against α-SMA showed many activated stellate cells (red), but their GFP expression was undetectable. GFP-positive cells were co-localizing hepatocytes, showing that GFP fluorescence was not diminished during cell fixation for α-SMA staining. (E, F) GFP expression in SECs incorporating only acetylated LDL (F, dotted area) or in Kupffer cells incorporating both acetylated LDL and latex beads (F, arrows) was also undetectable. GFP-positive cells here also indicated binuclear hepatocytes. Bars: (A, B) 50 μm, (C–F) 100 μm. Journal of Hepatology 2003 39, 162-170DOI: (10.1016/S0168-8278(03)00237-X)

Fig. 2 FCM of dissociated liver cells from selected GFP-transgenic mouse strain. SSCultra-low, SSClow, and SSChigh subpopulations were identified (upper). SSCultra-low cells (dotted circle), corresponding to hematopoietic cells, scantly expressed GFP, whereas SSChigh cells (thin circle), corresponding to mature hepatocytes, highly expressed GFP. Note that SSClow cells (solid circle) can be further fractionated into GFP−/low and GFPhigh subpopulations. Abbreviation: FSC; forward scatter, SCC; side scatter. Journal of Hepatology 2003 39, 162-170DOI: (10.1016/S0168-8278(03)00237-X)

Fig. 3 (A–F) Sequential observation of cell cluster formation by sorted cells. The figure shows findings of the phase contrast (A, C, E) and corresponding fluorescent images (B, D, F). Under low-density culture conditions, sorted cells attached to the culture dish in single and began to divide after 1 day of culture (A, B). The cells then formed a colony, after 3 days (C, D), and 7 days (E, F). (G) Immunocytochemistry for AFP after 7 days of culture. Cells forming colonies were AFP-positive immature endodermal cells. The small image shown lower left indicates a negative control image without primary antibody. (H) Western blotting for AFP. Lane 1; adult liver, Lane 2; fetal liver, Lane 3; cultured cell lysate from day 7. Bars: (A, B) 50 μm, (C–G) 100 μm. Journal of Hepatology 2003 39, 162-170DOI: (10.1016/S0168-8278(03)00237-X)

Fig. 4 (A, B) RT-PCR agarose gel electrophoretic images. (A) Results from cells before sorting; and (B) results from cells in colonies after 7 days of culture. All of the markers were positive in the pre-sorting condition. After 7 days of culture, the sorted cells expressed only endodermal markers, including AFP, albumin and CK19. (C) Quantitative analysis of mRNA expression by real-time PCR. In the post-sorting condition, endodermal markers including albumin, AFP and CK19 are the same or several times higher than the pre-sorting condition, whereas nearly complete elimination of non-endodermal cells was quantitatively confirmed by the almost zero level of desmin, CD45 and Flk1 expressions in sorted and cultured cells (n=3, *P<0.01). Journal of Hepatology 2003 39, 162-170DOI: (10.1016/S0168-8278(03)00237-X)

Fig. 5 Maturation and bilineage differentiation of the sorted cells. (A) Cultured sorted cells proliferated and formed colonies, however, they could not further expand and maturate without NPC feeder cells after 1 week of culture, and finally died after 4 weeks. (B) In contrast, in the case of culturing the sorted cells with NPCs from the GFP-negative strain as a feeder layer, predominant colonies began to express mature morphology. (C–H) Double immunocytochemical staining of 4-week cultured sorted cells co-cultured with GFP-negative non-parenchymal cells (E, H), shown with corresponding phase-contrast (C, F) and original GFP fluorescent image (D, G). Albumin and CK19 were marked by Alexa Fluor®350 (blue) and TexasRed®-X (red), respectively. (C, D, E) A colony with only albumin expression. Predominant colonies formed from sorted cells contained only albumin-positive cells as shown in Fig. 5E (arrows). (F, G, H) A colony with expression of both albumin and CK19. Purple-colored cells indicate albumin and CK19 double-positive cells (H, arrows). Some of them contain CK19 single-positive cells (H, arrowheads), or both albumin and CK19 positive cells (H, arrows). Bars: (A–H) 100 μm. Journal of Hepatology 2003 39, 162-170DOI: (10.1016/S0168-8278(03)00237-X)

Fig. 6 Electron microscopy of the sorted cells after long-term culture. Figures B and D are high magnified images of the boxed area in Figure A and C, respectively. (A, B) Most cells have numerous mitochondria, peroxisomes, tight junctions with desmosomes (arrows in B), and bile-canaliculi-like structures with microvilli (circle in B). (C, D) Cells constituting duct-like structures show a large nucleus-cytoplasm ratio, a lumen structure with short microvilli (arrowheads in D), and juxtaluminal junctional complexes (arrow in D). Original magnifications, ×2000 (A, C), ×4000 (B, D). Scale bars, 1 μm (A, C), 5 μm (B, D). Journal of Hepatology 2003 39, 162-170DOI: (10.1016/S0168-8278(03)00237-X)

Fig. 7 Analysis of specific surface markers using CD45−TER119−SSClowGFPhigh sorted cells. Known surface markers including c-Kit, Thy1.1 and integrin-α6 and -β1 were analyzed. The average percentage of positive cells for each marker was gained from five independent experiments (shown at lower right). Journal of Hepatology 2003 39, 162-170DOI: (10.1016/S0168-8278(03)00237-X)