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First published online 30 November 2004
doi: 10.1242/jcs.01572

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Long-term culture of hepatic progenitors derived from mouse Dlk⁺ hepatoblasts

¹ Stem Cell Regulation, Kanagawa Academy of Science and Technology (KAST), Teikyo University Biotechnology Research Center, 907 Nogawa, Kawasaki, Kanagawa 216-0001, Japan
² Department of Anatomy, Department of Biochemistry and Biophysics, University of California San Francisco, 600 16th street, San Francisco, CA 94143, USA
³ Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
⁴ Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Kawaguchi, 332-0012, Japan

View larger version (43K): [in a new window]   Fig. 1. Bi-directional differentiation of E14.5 Dlk+ cells. (A,B) Typical colonies formed from single Dlk+ cells on type IV collagen (A) or laminin (B) after 5 days of culture contain both CK19+ and albumin+ cells. Colonies were stained with anti-CK19 antibody and FITC-conjugated anti-rabbit IgG (green in panels 1), and anti-albumin antibody and Cy3-conjugated anti-goat IgG (red in panels 2). Panels 3 show the merged images of panels 1 and 2. Bar, 100 µm.

View larger version (45K): [in a new window]   Fig. 2. Dlk is significantly downregulated in culture. (A,B) Expression of Dlk was examined in colonies grown on type IV collagen (A, panel 2) and on laminin (B, panel 2) after 5 days of culture. Colonies were incubated with hamster anti-Dlk mAb and biotin-conjugated anti-hamster IgG followed by the treatment with FITC-conjugated streptavidin. The colonies were counterstained with hematoxylin to visualize nuclei (panels 1). Bar, 100 µm. (C) Expression of Dlk was examined by FACS. After 5 days of culture, cells dissociated from type IV collagen-coated plates were incubated with hamster IgG (panel 1) or anti-Dlk mAb (panel 2), and subsequently incubated with FITC-conjugated anti-hamster IgG.

View larger version (38K): [in a new window]   Fig. 3. Laminin supports long-term proliferation of Dlk+ cells. (A,B) The number of large colonies, containing more than 100 cells, observed at 5, 14 and 21 days after plating Dlk+ cells on type IV collagen (A) and laminin (B). While on type IV collagen, the number of large colonies decreased during long-term culture (A), some medium-sized colonies continued to expand on laminin after 5 days and became large colonies at 14 days (red portion of bar) and at 21 days (yellow portion of bar) (B). Large colonies were marked at the 5th day after plating and their sizes were checked at days 14 and 21. The colonies that contained more than 100 cells on day 14 and 21 were also marked. Data shown are the number of colonies formed from 1000 Dlk+ cells. The culture was repeated independently four times, and average values of colony number are shown. Error bars represent standard deviation for the total number of large colonies at each time point. Student's t-test was performed and P values are shown in A and B. (C) Expression of Ki67 after 21 days of culture. About 20% of the cells on laminin expressed Ki67 (panel 3), whereas most of the cells on type IV collagen did not (panel 2). Cultured cells were dissociated from plates after 21 days of culture, fixed in 4% paraformaldehyde, and permeabilized in methanol. After incubation with mouse IgG (panel 1) or mouse anti-human Ki67 mAb (panels 2 and 3) followed by treatment with FITC-conjugated anti-mouse IgG, the expression of Ki67 was examined by using FACScaliber. (D) The colony derived from a single Dlk+ cell grew continuously on laminin. The same colony was photographed at 5, 14, 21 and 31 days after plating. Bar, 200 µm.

View larger version (28K): [in a new window]   Fig. 4. Characteristics of HPPL. (A) HPPL strongly expressed a laminin receptor, integrin 6ß1. Expression of integrin 6 and ß1 was analyzed by FACScaliber using FITC-conjugated anti-CD29 (integrinß1) and PE-conjugated anti-CD49f (integrin 6) mAbs (panel 2). As a negative control, HPPL were incubated with FITC-conjugated hamster IgM and PE-conjugated rat IgG (panel 1). (B) HPPL did not express Dlk. HPPL were incubated with anti-Dlk mAb followed by FITC-conjugated anti-hamster IgG (panel 2). As a negative control, HPPL were incubated with hamster IgG and FITC-conjugated anti-hamster IgG (panel 1). (C) HPPL expressed both CK19 and albumin. HPPL detached form plates were mounted on glass slides by cytospinning and incubated with anti-CK19 serum and anti-albumin antibody. The expression of CK19 and albumin were visualized with Alexa 488 anti-rabbit IgG (green in panel 1) and Alexa 555 anti-goat IgG (red in panel 2), respectively. Panel 3 is a merged image of panels 1 and 2. Bar, 10 µm. (D) TGF and p57 were upregulated and downregulated, respectively, in HPPL when compared with expression in E14.5 Dlk+ hepatoblasts. Gene expression was examined by RT-PCR. The thermal cycle was repeated 30 times for TGF and GAPDH, and 35 times for p57.

View larger version (58K): [in a new window]   Fig. 5. Hepatic and cholangiocytic differentiation of HPPL. (A) Hepatic differentiation of HPPL was induced by OSM and EHS gel as shown by the expression of TAT and CPS (panel 1). The EHS gel treatment also induced the formation of cell clusters showing granulated cytosol and clear round nuclei (panel 3) when compared with the cells without OSM and EHS gel (panel 2). HPPL that became confluent were incubated with OSM for 5 days, and then overlaid with EHS gel for additional 5 days. OSM was not added to the medium during EHS-gel treatment. Numbers shown at the top of panel 1 represent days after addition of OSM. Bar, 25 µm. (B) Polysaccharide accumulation by HPPL. PAS staining showed that HPPL treated with OSM and EHS gel accumulated high levels of polysaccharide in their cytosol (red in panel 2) compared with HPPL before the induction of hepatic differentiation (panel 1). The nuclei were counter-stained with hematoxylin. Bar, 100 µm. (C) Clearance of ammonia from culture medium by HPPL. HPPL treated with OSM and EHS gel eliminated ammonium ions from the culture medium within 48 hours of incubation (open circles). By contrast, HPPL without hepatic differentiation failed to remove ammonium ions (filled circles). (D) Expression of cholangiocyte marker genes in HPPL before (lane 1) and after (lane 2) culture in collagen gel. HPPL were examined for the expression of CK7 and CK19, and integrin ß4 by RT-PCR after 1 week of culture in type I collagen gel. Mature hepatocytes and non-parenchymal cells (NPC) containing cholangiocytes isolated from adult liver were also examined for the expression of these genes. The thermal cycle was repeated 30 times for GAPDH and 35 times for CK7, CK19 and integrin ß4. (E) HPPL showed tube-like structures in collagen gel. After 5 days of incubation in type I collagen gel, the cells were stained with anti-CK19 antibody. Box in panel 1 was magnified and is shown in panel 2. Bar, 100 µm (panel 1); 20 µm (panel 2).

View larger version (26K): [in a new window]   Fig. 6. Pancreatic differentiation of HPPL. The expression of Pdx1, insulin, glucagon, and lipase was examined by RT-PCR. HPPL before pancreatic differentiation (lane 1) and HPPL depleted of dexamethasone (Dex) and added with retinoic acid (RA) (lane 2) were compared. Adult pancreas (lane 3) and liver (lane 4) were also examined for the expression of these genes as positive and negative controls, respectively. After HPPL became confluent, dexamethasone and insulin/transferrin/selenium (ITS) were eliminated from the culture medium. Then, HPPL were incubated for an additional 5 days with 2 µM RA. Gene expression was examined by RT-PCR. The thermal cycle was repeated 30 times for lipase and GAPDH, 35 times for Pdx1 and insulin, and 40 times for glucagon.

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