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First published online 4 March 2008
doi: 10.1242/jcs.019315

Journal of Cell Science 121, 1002-1013 (2008)
Published by The Company of Biologists 2008
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Phenotype reversion in fetal human liver epithelial cells identifies the role of an intermediate meso-endodermal stage before hepatic maturation

Mari Inada1,2,3, Antonia Follenzi1,2,4,5, Kang Cheng1,2,6, Manju Surana1,6, Brigid Joseph1,2, Daniel Benten1,2, Sriram Bandi1,2,8, Hong Qian7 and Sanjeev Gupta1,2,4,6,7,8,*

1 Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
2 Marion Bessin Liver Research Center, Ullmann Building 5th and 6th floors, 1300 Morris Park Avenue, Bronx, NY 10461, USA
3 Departments of Medicine and Clinical Oncology, Graduate School of Medicine, Chiba University, Japan
4 Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
5 University of Piemonte Orientale, Novara, Italy
6 Diabetes Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
7 Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
8 Center for Human Embryonic Stem Cell Research, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA


Figure 1
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  		Fig. 1. Gene expression in primary fetal liver cells following MACS. (a,b) Gene expression with two-color immunostaining in EpCAM-positive cells to demonstrate coexpression of Alb and CK-19 or vimentin (arrows). DAPI nuclear counterstain, blue color. Magnification: x200. (c) Gene expression by RT-PCR amplification in EpCAM-positive or -negative cell fractions, including hepatobiliary markers (Alb, CK-19), liver transcription factors (hepatic nuclear factors, HNFs) (lanes 1, 2), growth factors and receptors (lanes 3, 4) and cell-signaling molecules Wnt receptors, frizzled (FZ) and Notch (lanes 5, 6).

 

Figure 2
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  		Fig. 2. RT-PCR analysis of gene expression in Ep-CAM-positive cells. Gene expression in hESCs cultured under undifferentiating conditions (lane 1), EpCAM-positive fetal liver cells before cell culture (Pr, lane 2), during primary cell culture (P0, lanes 3, 5) and during the third passage in cell culture (P3, lanes 4, 6).

 

Figure 3
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  		Fig. 3. Generation of mesenchymal lineages from cultured fetal liver cells. P2 passage cells cultured under conditions to induce adipogenic and osteogenic differentiation. Cells cultured under standard conditions did not show staining for Oil Red O (a) or Alizarin (c). Under differentiation induction conditions, cells began to accumulate lipid droplets and acquired a foamy appearance (b), which is typically seen in adipocytes, or exhibited calcium deposition (d), which is typically seen in osteocytes. Magnification: x200.

 

Figure 4
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  		Fig. 4. Promoter activity in EpCAM-positive cells. P0 cells in early (4-7 day) intermediate (14-21 day) and late (30 day) cultures. (a) Cells with advancement along fibroblast-like morphology. (b-d) GFP expression in P0 cells transduced with lentiviral vector using PGK, Alb and TTR promoters. (e,f) Late P0 cultures with CK-19 staining (e, red color) and merged image showing coexpression of GFP under TTR promoter (f, green color=GFP; arrows indicate CK-19-GFP coexpressing cells). (g,h) Epithelial cells in P3 cultures with merged images of CK-19 immunostaining and GFP under either TTR (g) or Alb promoter (h) (arrows). DAPI nuclear counterstain (blue). Magnification: x200.

 

Figure 5
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  		Fig. 5. Analysis of clonal EpCAM-positive cells. (a-c) Freshly isolated cells under phase (a), after GGT staining, red color, with hematoxylin counterstaining, blue color (b) and after glycogen staining (c). (d) An expanded colony of clonal P1 cells after culture for 6 weeks. (e-i) Cytospun preparations of cells released from late P1 culture with GGT (e) glycogen (f), as well as vimentin (g-i) staining in cells. The data verified that clonal cultured cells maintained epithelial characteristics while acquiring additional mesenchymal properties. Magnification: x200.

 

Figure 6
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  		Fig. 6. Meso-endodermal phenotype in fetal liver cells. (a) Immunostaining of early P0 culture (7 days) with GFP lentiviral vector expression under TTR promoter (left, green color), filamentous vimentin (middle, red color) and merged image (on right) showing coexpression in cells of GFP and vimentin. (b) Similar findings in late P0 culture, with coexpression of GFP lentiviral vector under Alb promoter and filamentous vimentin, except for morphological alterations. (c) P7 culture with GFP expression under Alb promoter, filamentous {alpha}-SMA expression (middle) and merged image showing cells with either or both properties (right). DAPI nuclear counterstain. Magnification: x200. (d) Summary of gene expression switches with quantitative RT-PCR showing mean fold-change in mRNA abundance in freshly isolated fetal liver cells (primary), early P0 culture (7 d) and multi-week P3 cultures. Alb, AFP, CK-19, E-cadherin, and HNF1 and HNF4 were expressed less in P0 and P3 cells, while HNF3{alpha} (FOXA1) expression reappeared. By contrast, expression of vimentin, {alpha}-SMA, TGFβ1 and TGFβ2 and receptors, increased. Data were normalized against β2-microglobulin. An arbitrary baseline was assigned to HNF3{alpha}.

 

Figure 7
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  		Fig. 7. Microarray analysis of gene expression in fetal liver cells. (a) Volcano plots of gene expression in freshly isolated cells (PP) vs P0, P1 or P3-P6 cultures. Upper right and left regions show >twofold and statistically significant differences in gene expression (green=down, red=up). (b) Overall distribution of differentially expressed genes and fractions representing two gene ontology groups and KEGG pathways. (c) Identification of genes expressed in various cell populations. The data were from total gene sequences considered to have been expressed, range, 44,256 to 50,319. Compared with PP cells, 191 genes were uniquely downregulated in P0 and P1 and 177 genes were uniquely downregulated in P3-P6. By contrast, 712, 302 and 363 genes were upregulated in P0, P1 and P3-P6, respectively. Changes in TGFβ signaling (d) and BMP signaling (e) in cultured cells compared with PP cells. Genes with higher expression in PP are in pink, those expressed less well in PP are in green, and those not represented are in grey. In PP cells, Smad2 (MADH2) and Smad3 expression was consistent with their positive roles in endoderm development and hepatic differentiation, while Smad7 (MADH7, the negative regulator of Smad2 and Smad3) was expressed simultaneously, and Smad4 and ERK activator kinase were less active. By contrast, during subsequent cell culture, Smad7 inhibition was removed, along with downregulation of ERK activator kinase and Ski-like (SKIL), which inhibit the Smad signaling regulating endodermal differentiation. Analysis of BMP signaling (e) indicated that Smad4 was expressed more highly in P0 cells and BMP2 was expressed more highly in P3-P6 cells. Depending on the context of Smad4 activity, BMP2 signaling can favor mesoderm or endoderm induction. Taken together, perturbations in TGFβ and BMP signaling were in agreement with the fetal cell phenotypes observed.

 

Figure 8
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  		Fig. 8. Regulation of meso-endodermal phenotype. Perturbations in P3 fetal liver cells cultured with FBS (control) or hHGF, OSM and no serum. (a) Cell morphology under phase contrast with more compact cells in OSM or no serum. (b) Vimentin staining (green color). (c) {alpha}-SMA staining (red color) with characteristic filamentous pattern seen in mesenchymal cells and less pronounced expression in cells cultured with no serum (panel on extreme right). (d,e) Cells transduced with GFP lentiviral vector under PGK or Alb promoter, respectively. While PGK expression remained unaltered, Alb promoter was more active in cells cultured with OSM or no serum. Magnification: 200x. (f) Flow cytometry analysis indicating twofold and fourfold greater mean fluorescence intensity (MFI) of GFP expression under Alb promoter in cells cultured with OSM or no serum. (g) Changes in gene expression in P3 cells with qRT-PCR using β2-microglobulin as internal control. Vimentin expression declined to 60% of controls and {alpha}-SMA expression declined to 72% and 26% of controls in cells cultured with OSM or no serum, respectively. In cells without serum, HNF-3{alpha} (FOXA1) expression increased.

 

Figure 9
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  		Fig. 9. EM analysis of cellular ultrastructure. (a) Freshly isolated PP cells with large nuclei, prominent nucleoli and relatively sparse cytoplasm with scattered mitochondria (m) and secondary lysosomes (Ly). P0 cells (b) and P3 cells (c) that had been in culture for three weeks and seven weeks, respectively. These cells showed intermediate filaments (F, arrows) as well as complex cytoplasmic appearance with multiple mitochondria, vacuoles, occasional primary lysosomes (P Ly) as well as secondary lysosomes and microperoxisomes (P) consistent with alterations in cell morphology and presence of both mesenchymal and epithelial features. (d) P3 cells cultured without serum. Under this condition, most cells no longer contained intermediate filaments, although the epithelial phenotype did not resemble that of freshly isolated PP cells, indicating the incompleteness of phenotypic reversion. These cells again contained primary and secondary lysosomes as well as microperoxisomes, which are additionally shown in insets. Scale bars: 2 µm.

 

Figure 10
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  		Fig. 10. Changes in OSM signaling. (a-c) Microarray data analysis of OSM signaling in PP cells versus cultured cells. Genes with higher expression in PP cells are in pink, those expressed less well in PP cells are in green, those not expressed are in grey (IL6ST, interleukin-6 signal transducer). The OSM signaling pathway was intact, although STAT3 was upregulated in cultured cells, probably explaining the responsiveness of P3 cells to added OSM.

 

Figure 11
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  		Fig. 11. Fate of fetal cells in vivo. (a) Transplanted cells in the peritoneal cavity or liver (lanes 2, 3) of immunodeficient mice with PCR probe identifying CMT1A human sequences. (b) In situ hybridization signals with human pancentromeric probe using FITC-conjugated anti-digoxigenin in human liver. (c) No hybridization signals with this probe were seen in mouse liver. (d) Histochemical staining of human liver for glycogen (pink color in cytoplasm) followed by in situ hybridization with human pancentromeric probe using peroxidase-conjugated anti-digoxigenin and development with diaminobenzidine (dark brown color in nuclei). (e,f) Transplanted fetal human cells (arrows) in the peritoneal cavity or liver (f) using human in situ hybridization probe. mc, microcarriers; Pa, portal area. (g) Immunostaining for human albumin in a transplanted cell (arrow) in the liver. (h-j) Transplanted cells (arrows) transduced with GFP lentiviral vector containing PGK, Alb or TTR promoters, with GFP immunostaining (green) and DAPI nuclear stain (blue). (k,l) Histochemical staining for glycogen (pink) followed by in situ hybridization using human pancentromeric probe to verify that lentiviral-vector-transduced cells (brown nuclear signals, arrows) were hepatocytes. (m-p) The same liver area verifying absence of fusion in lentiviral-vector-transduced human cells and native mouse cells with in situ hybridization using human plus mouse pancentromeric probes. A transplanted cell (arrow, m) hybridizes with the human probe. Hybridization signals with the mouse probe in cells other than the human cell (n), DAPI staining of nuclei (o) and merged view of m-o (p) to indicate that mouse signals were absent in the transplanted human cell and vice versa. Magnification: x400 (b-f, h-k); x600 (g,l, m-p).

 

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© The Company of Biologists Ltd 2008