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First published online 11 March 2003
doi: 10.1242/jcs.00388

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Isolation of hepatoblasts based on the expression of Dlk/Pref-1

¹ Stem Cell Regulation, Kanagawa Academy of Science and Technology (KAST), Teikyo University Biotechnology Research Center, 907 Nogawa, Kawasaki, Kanagawa 216-0001, Japan
² Kirin Pharmaceutical Research Lab, Takasaki, Gunma 370-1295, Japan
³ Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
⁴ First Department of Pathology, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan

View larger version (63K): [in a new window]   Fig. 1. Expression of Dlk mRNA in E10.5 embryo detected by in situ hybridization. E10.5 mouse embryo fixed in 4% PFA was hybridized with DIG-labeled sense (A) and antisense (B) probes of Dlk as described in Materials and Methods. After incubation with alkaline phosphatase (AP)-conjugated anti-DIG antibody, the signal was visualized by AP activity using BCIP/NBT as a substrate. Dlk was detected in the liver bud, which is seen under the heart, as well as vertebra. flb, forelimb bud; h, heart; lb, liver bud.

View larger version (37K): [in a new window]   Fig. 2. Expression of Dlk in immature liver cells in vivo and in vitro. RT-PCR (A) and northern blotting (B,C) were performed to detect Dlk mRNA during liver development and fetal hepatocyte primary culture. (A) Dlk expression was clearly detected in the E10.5 liver bud and the E14.5 liver but not in the neonatal liver shown by RT-PCR using cDNA synthesized from total RNA of livers at each stage. GAPDH expression was also examined to ensure an equal quantity of cDNA used for PCR. (B) Dlk was strongly expressed in fetal livers between E12.5 and E16.5, but it was rapidly downregulated in later gestation. In neonatal and adult livers, its expression was not detected. Each lane was loaded with 10 µg of total RNA extracted from livers at each stage. GAPDH expression was also examined to ensure an equal loading of RNA. (C) Fetal hepatic cells were prepared from E14.5 fetal liver and cultured on gelatin-coated dishes in the presence or absence of dexamethasone (Dex) and oncostatin M (OSM). TAT and CPS were weakly expressed after 4 days and clearly detected after 7 days of culture with Dex and OSM. Each lane was loaded with 10 µg of total RNA extracted from cultured cells at each time point. Note that Dlk was rapidly downregulated in the presence of Dex and OSM, although downregulation occurred spontaneously without OSM.

View larger version (151K): [in a new window]   Fig. 3. Histochemical analysis of Dlk expression in mouse liver. (A-D) Horizontal (A,B,D) and sagittal (C) sections of frozen E10.5 embryo were stained with anti-Dlk mAb. Dlk was detected in the E10.5 liver bud, but not in gut tubes, heart and forelimb bud (A-C). Higher magnification of the box in B is shown in D. The endodermal cells of the liver bud were stained with anti-Dlk mAb, whereas those of the gut tube were not stained. (E,F) E14.5 liver (E) and adult liver (F) were also incubated with anti-Dlk mAb. Dlk was expressed in E14.5 fetal liver but not in adult liver. (G-J) E14.5 hepatic cells were mounted on glass slides and incubated with anti-Dlk mAb and anti albumin antibody. Cell nuclei were stained with hematoxylin (G). The immunofluorescence staining of Dlk and albumin was visualized with FITC (H) and rhodamine (I), respectively. Large fetal hepatic cells (arrowheads in G) were stained with anti Dlk mAb (green in H) and anti-albumin (red in I). Dlk+ cells were identical to albumin+ cells (yellow in J). Dlk- cells with large nuclei and less cytoplasm were mostly hematopoietic cells. (K-M) Continuous frozen sections of E17.5 fetal liver were stained with anti-Dlk mAb (K), anti-CK19 antibody (L), and both antibodies (M). Dlk+ cells (brown in K) and CK19+ biliary epithelial cells (blue in L) were completely distinguishable (M). CK19+ bile ducts were visible around portal veins as well as ductal plates consisting of double layers of CK19+ cells. bd, bile duct; fg, foregut; flb, forelimb bud; h, heart; lb, liver bud; mg, mid-gut; nt, neural tube; pv, portal vein. Bars, 100 (A-D); 50 µm (E-M).

View larger version (48K): [in a new window]   Fig. 4. Flow cytometric analysis of Dlk expression and isolation of Dlk+ cells. (A) E14.5 liver cells were stained with anti-Dlk mAb and analyzed by FACS. Dlk+ cells were separated from Dlk- cells, which mainly consisted of hematopoietic and endothelial cells. Dlk+ cells were about 10% of total E14.5 hepatic cells. (B) Dlk+ cells isolated by AutoMACS were stained with anti-AFP (1) and anti-albumin antibodies (2). The morphologies of Dlk+ cells are shown in the upper panels. Over 95% of Dlk+ cells were stained with anti-AFP and anti-albumin antibodies (red in lower panel). (C) cDNA was synthesized from total RNA of CD45-TER119-Dlk- and CD45-TER119-Dlk+ cells separated by FACSvantage. Quantitative PCR reaction was performed with a LightCycler. The signals for AFP and albumin were normalized with the GAPDH level and relative expression level (the expression level in Dlk- cells = 1) are shown. The mRNA levels for AFP and albumin in Dlk+ cells (white bar) were 60 and 40 times, respectively, those in Dlk- cells (black bar).

View larger version (17K): [in a new window]   Fig. 5. Colony formation of E14.5 Dlk- and Dlk+ cells. Dlk- and Dlk+ cells were isolated from E14.5 fetal liver by AutoMACS and cultured at a density of 1000 and 50 cells/cm2, respectively, on type IV collagen-coated 6-well plates in the presence of 20 ng/ml of HGF and EGF. The number of cells in each colony was counted after 5 days of culture. Data shown here are numbers of colonies formed from 500 Dlk- (black bar) and Dlk+ (white bar) cells. 24±3% of E14.5 Dlk+ cells formed various sizes of colony after 5 days of culture. Among those colony-forming Dlk+ cells, 10% formed large colonies containing over 100 cells.

View larger version (103K): [in a new window]   Fig. 6. Colonies formed from E14.5 Dlk+ cells consist of albumin+ and CK19+ cells. (A-C) Dlk+ cells were isolated from E14.5 fetal liver by AutoMACS and cultured on chamber slides coated with type IV collagen at a density of 50 cells/cm2 in the presence of HGF and EGF. After 5 days of culture, cells were fixed and stained with anti-albumin and anti-CK19 antibodies. Expression of albumin and CK19 was visualized with Cy-3 and FITC, respectively. Various sizes of colonies (A, a small colony containing about 40 cells; B, a medium colony containing about 60 cells; C, a large colony containing over 100 cells) were formed in a low density culture, which contained both albumin+ (red in A-1, B-1 and C-1) and CK19+(green in A-2, B-2 and C-2) cells. Both images were merged in A-3, B-3 and C-3. (D) A large colony formed from Dlk+ cells was picked from the culture dish and mounted on a glass slide by cytospin. The cells were stained with anti-albumin and anti-CK19 antibodies. Some of them expressed both albumin (red in D-1) and CK19 (green in D-2). In D-3, both images were merged. (E) A single Dlk+ cell was isolated and inoculated in one well of a 96-well plate coated with type IV collagen by FACSvantage. A large colony derived from a single Dlk+ cell consisted of albumin+ (red in E-1) and CK19+ (green in E-2) cells. Both images were merged in E-3. Bars, 100 µm.

View larger version (68K): [in a new window]   Fig. 7. Electron microscopic analysis of Dlk+ cells before and after the culture. Dlk+ cells isolated from E14.5 fetal liver and the cells collected from the low density culture were fixed and used for preparing thin sections for transmission-electron microscopy. (A) E14.5 Dlk+ cells exhibited a round-shaped nucleus, small nucleolus and elongated mitochondria. (B) Cells cultured at low density showed characteristics distinct from freshly isolated cells. A typical type of cells is shown. There are many microvilli on the surface, a cleaved nucleus, larger nucleolus, and many round-shaped mitochondria in the cytoplasm. Bars, 2 µm.

View larger version (43K): [in a new window]   Fig. 8. Engraftment of Dlk+ cells in recipient liver. E14.5 Dlk+ cells isolated from GFP transgenic mice were transplanted intrasplenically into recipient mice injured by intraperitoneal administration of anti-Fas antibody, Jo2. Thirty six weeks after transplantation, frozen sections of recipient liver were made. (A) GFP+ cells were detected in liver parenchyma (green). Higher magnification of the box in A is shown in B. (C) The frozen section was stained with anti-albumin antibody. GFP+ cells expressed albumin (red in C). (D) Overlay image of GFP, albumin (red), and DAPI (blue).