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doi: 10.1242/10.1242/dev.00459

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Role for growth factors and extracellular matrix in controlling differentiation of prospectively isolated hepatic stem cells

¹ Department of Surgery, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
² Laboratory of Stem Cell Therapy, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
³ Department of Regenerative Medicine, Faculty of Medicine, Yokohama City University, Yokohama, Kanagawa 236-0004, Japan

View larger version (20K): [in a new window]   Fig. 1. Experimental procedure to elucidate the manner of hepatic stem cell differentiation. The Met+ CD49f+/low Kit– CD45– TER119– cells isolated from E13.5 fetal mouse livers were cultured clonally in 96-well plates. Cells that expanded in culture and produced hepatocytes and cholangiocytes as descendants while maintaining primitive stem cells undergoing self-renewing divisions were used to analyze differentiation status. We chose three independent stem cell clones for examination. Following transfection of the ALB enhancer/promoter-EGFP construct, cells were examined by using FACS and quantitative PCR after they had been cultured with several GFs or ECMs. Scale bar: 100 µm.

View larger version (43K): [in a new window]   Fig. 2. Separation of differentiating ALB+ cells from the stem cell population. (A) After gene transfer of the ALB enhancer/promoter-EGFP construct into expanding stem cell populations, ALB+ and ALB– cells were sorted restrictively using FACS. Immediate re-analysis of sorted cells showed purification of both ALB+ and ALB– cells. After the culture of each sorted cell subpopulation for 16 days on type IV collagen-coated dishes, FACS analysis demonstrated that ALB– cells emerged from ALB+ cells, and that ALB+ cells emerged from ALB– cells. Percentages of fractionated cells are shown at the top of each panel. Establishment of the gate was based on the profile of the negative control. (B) Semi-quantitative RT-PCR analysis of sorted EGFP+ (ALB+) and EGFP– (ALB–) cells. Note that ALB+ sorted cells expressed hepatocyte-lineage markers, such as ALB, At, G6P and TO, at much higher levels than did ALB– cells. (C) Quantitative analysis of sorted ALB+ cells using real-time quantitative PCR. All data were normalized to the value of ALB– sorted cells and fold-differences are shown. Representative data from a transfected stem cell clone are shown; three samples were examined for each protein. Data are mean±s.d. (D) (a-f) Several sorted ALB– cells could form clonal colonies including both ALB+ and ALB– cells at day 20. (g-i) Moreover, ALB+ sorted cells gave rise to ALB– cells even 1 day after the initiation of culture, and finally formed mosaic colonies similar to those from ALB– cells (black arrowhead in g, an original EGFP+ cell; white arrowhead in g, a daughter cell). (a,d,g) Phase contrast. (b,e,h) Enhanced green fluorescence protein (EGFP) imaging. (c,f,i) Merge. d-f are magnifications of a-c, respectively. Scale bars: 100 µm.

View larger version (20K): [in a new window]   Fig. 3. Growth and differentiation of sorted ALB– and ALB+ cells cultured with GFs and ECMs. FACS-sorted ALB– and ALB+ cells were cultured separately. For the GF test, cells were cultured with or without HGF, OSM, aFGF, bFGF or a mixture of all GFs on type IV collagen-coated dishes. For the ECM test, cells were cultured on either non-coated, or laminin-, type IV collagen-, type I collagenor fibronectin-coated dishes. After 10 days in culture, proliferation (A) and differentiation (B) of sorted cells were examined by cell counting (A) or FACS (B). Representative data from a transfected stem cell clone are shown; three samples were examined for each GF or ECM. Data are mean±s.d. (A) HGF strongly promoted the proliferation of both ALB– and ALB+ sorted cells, whereas lesser effects were noted with OSM, aFGF and bFGF. OSM specifically suppressed the proliferation of ALB– cells. For the ECMs, laminin, type IV collagen and type I collagen induced the proliferation of ALB+ cells, but to a lesser degree than HGF. All data were normalized to the value of no-GF (GF test) or a non-coated dish (ECM test) and fold-differences are shown. (B) FACS-analysis revealed that HGF and OSM have the potential to induce ALB+ cells from ALB– sorted cells, and inhibit generation of ALB– cells from ALB+ sorted cells. Laminin, type IV collagen and type I collagen possessed similar, but lesser, effects than did HGF and OSM. All data are normalized to the value at the day of sorting (day 0) (GF test) or to the value of a non-coated dish (ECM test) and fold differences are shown.

View larger version (12K): [in a new window]   Fig. 4. Quantitative analysis of the effect of HGF and OSM on sorted ALB– and ALB+ cells. FACS-sorted ALB– and ALB+ cells were cultured separately with or without HGF or OSM on type IV collagen-coated dishes for 10 days. Then, quantitative PCR was performed to determine the expression of several hepatocyte or cholangiocyte marker genes. Representative data from a transfected stem cell clone are shown; three samples were examined for each GF. Data are mean±s.d. (A) In the ALB– cell culture, HGF strongly induced ALB and At expression. The mid-latter marker G6P was induced exclusively by OSM, but TO expression was still not detected. In the ALB+ cell culture, however, OSM strongly induced G6P and TO expression. The expression of ALB in the ALB+ sorted cell cultures was also promoted by HGF and OSM, but its effects were not greater than in ALB– cells. (B) Both HGF and OSM suppressed the expression of cholangiocyte marker genes such as CK19 and GGT in ALB– and ALB+ sorted cell cultures. All data were normalized to the values of no-GF and fold differences are shown. ND, not detected.

View larger version (16K): [in a new window]   Fig. 5. HGF and several ECMs regulate C/EBP expression during stem cell differentiation. (A) When cells were cultured on type IV collagen-coated dishes for 8 days, C/EBP expression was promoted in a dose-dependent manner by HGF, as well as by the presence of laminin, type IV collagen and type I collagen. By contrast, C/EBPß expression was decreased in these culture conditions. All data were normalized to the expression values from 0 ng/ml (HGF) or non-coated dishes (ECM) and fold differences are shown. Representative data from a stem cell clone are shown; three samples were examined for each concentration of HGF or each ECM. Data are mean±s.d. (B) C/EBP was highly expressed in ALB– and ALB+ sorted cells cultured with HGF for 10 days. Its expression was promoted exclusively in the transition of ALB– cells to ALB+ hepatic precursors induced by HGF. All data were normalized to the value of no-GF and fold differences are shown. Representative data from a transfected stem cell clone are shown; three samples were examined for each GF. Data are mean±s.d.

View larger version (17K): [in a new window]   Fig. 6. Dominant-negative C/EBP (A-C/EBP) inhibits hepatocyte differentiation from stem cells. (A,B) After culturing FACS-sorted EGFP+ transduced cells for 10 or 20 days, quantitative PCR was performed. The expression of hepatocyte-differentiation markers ALB, AFP, At, G6P and TO in the stem cell population was significantly suppressed even when cultured with both HGF (H) and OSM (O), which induce hepatocyte differentiation. By contrast, the expression of CK19 and GGT, which are markers of cholangiocyte differentiation, was elevated in cells expressing A-C/EBP. The expression of HNF1, HNF4 and Met was also relatively enhanced, but not significantly. The expression of HNF6, however, was decreased in transduced cells, in a similar fashion to hepatocyte marker genes. All data were normalized to the value of mock controls (day 10; no GF) and fold differences are shown. Representative data from a stem cell clone are shown; three samples were examined for each condition. Data are mean±s.d. (C) Immunocytochemical staining of ALB and PAS-staining were performed on cells expressing EGFP (mock) or A-C/EBP at day 20. In cells expressing A-C/EBP, few cells were positive for ALB and stored glycogen. (counterstain: Hematoxylin). (D) After culture of sorted EGFP (mock) or A-C/EBP-expressing cells for 5 days in clonal density cultures (5x102 cells/well in six-well plates), the number of large (>100 cells in a colony) and small colonies was counted (n=6). The number of large colonies was increased when cells were transduced with A-C/EBP. Data are mean±s.d. (*P<0.005). (E) After transduction of FACS-sorted ALB+ or ALB– cells by A-C/EBP-NGFR and culture of each cell population for 14 days, ALB+/NGFR+ cells or ALB–/NGFR+ cells were sorted separately and cultured with or without HGF and OSM for 10 days (plating density is 1x103 cells/cm2). Then, the generation of ALB– cells from ALB+/NGFR+ sorted cells and the generation of ALB+ cells from ALB–/NGFR+ sorted cells was examined by using FACS. Compared with mock controls, cells expressing A-C/EBP failed to maintain ALB expression in ALB+ sorted cells and were unable to generate ALB+ cells from ALB– sorted cells. Representative data from a transfected stem cell clone are shown; three samples were examined for each condition. Data are mean±s.d. Scale bars: 100 µm.

View larger version (34K): [in a new window]   Fig. 6. Dominant-negative C/EBP (A-C/EBP) inhibits hepatocyte differentiation from stem cells. (A,B) After culturing FACS-sorted EGFP+ transduced cells for 10 or 20 days, quantitative PCR was performed. The expression of hepatocyte-differentiation markers ALB, AFP, At, G6P and TO in the stem cell population was significantly suppressed even when cultured with both HGF (H) and OSM (O), which induce hepatocyte differentiation. By contrast, the expression of CK19 and GGT, which are markers of cholangiocyte differentiation, was elevated in cells expressing A-C/EBP. The expression of HNF1, HNF4 and Met was also relatively enhanced, but not significantly. The expression of HNF6, however, was decreased in transduced cells, in a similar fashion to hepatocyte marker genes. All data were normalized to the value of mock controls (day 10; no GF) and fold differences are shown. Representative data from a stem cell clone are shown; three samples were examined for each condition. Data are mean±s.d. (C) Immunocytochemical staining of ALB and PAS-staining were performed on cells expressing EGFP (mock) or A-C/EBP at day 20. In cells expressing A-C/EBP, few cells were positive for ALB and stored glycogen. (counterstain: Hematoxylin). (D) After culture of sorted EGFP (mock) or A-C/EBP-expressing cells for 5 days in clonal density cultures (5x102 cells/well in six-well plates), the number of large (>100 cells in a colony) and small colonies was counted (n=6). The number of large colonies was increased when cells were transduced with A-C/EBP. Data are mean±s.d. (*P<0.005). (E) After transduction of FACS-sorted ALB+ or ALB– cells by A-C/EBP-NGFR and culture of each cell population for 14 days, ALB+/NGFR+ cells or ALB–/NGFR+ cells were sorted separately and cultured with or without HGF and OSM for 10 days (plating density is 1x103 cells/cm2). Then, the generation of ALB– cells from ALB+/NGFR+ sorted cells and the generation of ALB+ cells from ALB–/NGFR+ sorted cells was examined by using FACS. Compared with mock controls, cells expressing A-C/EBP failed to maintain ALB expression in ALB+ sorted cells and were unable to generate ALB+ cells from ALB– sorted cells. Representative data from a transfected stem cell clone are shown; three samples were examined for each condition. Data are mean±s.d. Scale bars: 100 µm.

View larger version (20K): [in a new window]   Fig. 7. Summarized possible mechanism for hepatic stem cell differentiation. Because early-generated ALB+ cells can proliferate exclusively and differentiate into cholangiocyte-lineage cells, we speculate that transitory hepatic precursors expressing ALB exist in the developing liver and they are equivalent to bi-potent hepatoblasts.