First published online May 8, 2009
doi: 10.1242/10.1242/dev.031369
Development 136, 1951-1960 (2009)
Published by The Company of Biologists 2009
Potential hepatic stem cells reside in EpCAM+ cells of normal and injured mouse liver
Mayuko Okabe1,*,
Yuko Tsukahara1,*,
Minoru Tanaka1,*,
,
Kaori Suzuki1,
Shigeru Saito1,
Yoshiko Kamiya1,
Tohru Tsujimura2,
Koji Nakamura3 and
Atsushi Miyajima1
1 Laboratory of Cell Growth and Differentiation, Institute of Molecular and
Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan.
2 Department of Pathology, Hyogo College of Medicine, Nishinomiya, Hyogo
663-8501, Japan.
3 LivTech, Miyamae-ku, Kawasaki, Kanagawa 216-0001, Japan.
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Fig. 1. Expression profiles of candidate genes in normal and injured mouse and
rat liver. (A) Northern blot analysis of candidate genes in mouse
liver. The expression of these genes was selectively upregulated in DDC liver,
but not in injured liver without oval cell activation. (B) Quantitative
RT-PCR of Epcam and Trop2 in rat liver. Whereas
Epcam was expressed in normal rat liver (cont.) and upregulated in
2-AAF/partial hepatectomy (PH)-treated liver, Trop2 was not expressed
in normal liver but was expressed in 2-AAF/PH-treated liver. N, adult mouse
normal liver; O, DDC liver (6 weeks); P, liver 48 hours after 70% PH; C, liver
24 hours after carbon tetrachloride administration.
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Fig. 2. DDC diet causes hepatic injury and oval cell activation. (A)
The liver turned black after mice were fed a DDC diet. (B) H&E
staining of a frozen section of mouse normal liver (top) and 4 weeks after DDC
feeding (middle and bottom). Numerous small cells appeared around the portal
veins in the DDC liver (arrows). The brown clots represent the deposition of
iron hemes (arrowheads). (C) Immunohistochemistry (IHC) with anti-CK19
antibody showed that these numerous small cells included CK19-expressing oval
cells (arrows) in DDC liver. PV, portal vein. Scale bars: 100 µm
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Fig. 3. EpCAM is a cell surface marker for mouse oval cells. (A) IHC
of frozen liver sections with anti-EpCAM antibody after DDC feeding. EpCAM was
expressed in cholangiocytes around the portal vein of normal mouse liver (0
w). Feeding DDC caused the proliferation of EpCAM+ cells (1 and 4
weeks). (B) IHC of frozen liver sections with anti-EpCAM and anti-CK19
antibodies after 4 weeks of DDC feeding. (C) Flow cytometry (FCM) of
non-parenchymal cells (NPCs) prepared from the liver of mice fed DDC for 4
weeks with anti-EpCAM antibody and either CD45 or PECAM antibody.
EpCAM+ cells were negative for CD45 (hematopoietic marker) and
PECAM (endothelial marker). (D) Immunostaining of EpCAM+
cells with anti-A6 and anti-CK19 antibodies by cytospin. EpCAM+
cells expressed both molecules. (E) Immunostaining of EpCAM+
cells sorted from normal and DDC liver with anti-Ki67 antibody by cytospin.
Many EpCAM+ cells from DDC liver were stained with Ki67
(arrowheads). (F) The percentage of Ki67+ cells among
EpCAM+ cells after DDC treatment. The data are derived from five
different fields of view. Error bars, s.d. PV, portal vein. Scale bars: 100
µm.
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Fig. 4. Expression of EpCAM and TROP2 in normal and injured mouse liver.
(A,B) IHC of frozen sections of normal liver (A) and the liver
of mice fed DDC for 5 weeks (B) with anti-EpCAM and anti-TROP2 antibodies.
TROP2 was expressed in oval cells but not in normal cholangiocytes. (C)
FCM of NPCs with anti-EpCAM and anti-TROP2 antibodies after DDC feeding. TROP2
begins to be expressed in EpCAM+ cells as DDC feeding proceeds. PV,
portal vein. Scale bars: 100 µm.
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Fig. 5. Characterization of freshly isolated EpCAM+ cells.
(A) RT-PCR of freshly isolated EpCAM+ and EpCAM-
cells from the liver of mice fed DDC for 4 weeks. NPCs from DDC liver were
divided into EpCAM+ and EpCAM- cells by FACSVantage,
then RT-PCR was performed. (B) FCM of EpCAM+ cells from
normal and DDC livers with known oval cell markers. EpCAM+ cells
surrounded by bold lines were reanalyzed with other antibodies (TROP2, CD133,
CD34, c-KIT, THY1) as shown.
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Fig. 6. EpCAM+ cells derived from DDC liver have high proliferative
potential. (A,B) In vitro culture of EpCAM+
cells. Freshly isolated EpCAM+ cells from DDC liver were seeded on
type-I collagen-coated dishes in the presence of HGF, EGF and IL6. The
morphology of the cells after 5 days of culture (A) and after several passages
(B) is shown. (C) RT-PCR of EpCAM+ cells after 30 days of
culture. Afp was strongly expressed in the cultured cells. (D)
Immunostaining of the cultured cells with anti-CK19 and anti-ALB
antibodies.
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Fig. 7. Clonally expanded HSCEs can differentiate into both hepatocytes and
cholangiocytes. (A) Experimental design for the differentiation of
hepatic stem-like cells derived from EpCAM+ cells (HSCEs) into
hepatocytic cells. (B) RT-PCR of clone HSCE1 after hepatocytic
differentiation. The addition of OSM, DMSO and EHS gel strongly induced the
expression of hepatocytic genes and downregulated that of hepatoblastic and
cholangiocytic genes. (C) PAS staining of HSCE1. The addition of OSM,
DMSO and EHS gel strongly induced the accumulation of glycogen. (D)
Morphological changes of HSCE1 after cholangiocytic differentiation. Tubules
and branching morphology were clearly observed after 11 days of culture.
(E) RT-PCR of HSCE1 after cholangiocytic differentiation. The
expression of cholangiocytic marker genes was markedly upregulated in
HSCE1.
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Fig. 8. Comparison between EpCAM+ cells isolated from normal and
injured liver. (A) Colony formation assay of EpCAM+
cells from normal liver. Representative morphology of a small colony (top) and
a large colony (bottom). Large colonies composed of more than 100 cells after
9 days of culture proliferate exponentially. (B) Unlimited cell
proliferation of the established clones (#1-#4) in in vitro culture.
(C) Comparison of cell surface markers between HSCEs from normal (blue
line) and injured (red line) liver by FCM. Control IgG is in gray. The
expression profiles of cell surface markers were similar in both HSCEs.
(D) The number of EpCAM+ cells per normal (n=4) or
injured (n=6) liver. The number was estimated from the percentage of
EpCAM+ cells after immunomagnetic bead selection (see Fig. S2 in
the supplementary material). There was a significant increase in
EpCAM+ cells in DDC liver (*P<0.01).
(E) Colony formation assay of EpCAM+ cells from normal and
injured liver of mice fed DDC for 4 weeks. The data are derived from four
independent experiments. Error bars, s.d.
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Fig. 9. Model of the hepatic stem/progenitor cell system in vivo and in
vitro. Potential HSCs exist in normal liver as EpCAM+ cells.
They can proliferate unlimitedly and differentiate into both hepatocytes and
cholangiocytes in vitro. Upon liver injury without oval cell activation,
hepatocytes proliferate and contribute to liver regeneration. Upon liver
injury with oval cell activation, EpCAM+ TROP2+ cells
appear around portal veins to regenerate the liver. The oval cells might be
partly derived from EpCAM+ cholangiocytes. Most oval cells lacked
the potential to self-renew in the in vitro colony formation assay.
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© The Company of Biologists Ltd 2009
