studies

546A AASLD ABSTRACTS HEPATOLOGY, October, 2015
However, the potential of hepatocytes to stably and functionally
contribute to the biliary system is uncertain. Here, we use a
mouse model of Alagille syndrome (ALGS) that lacks peripheral
intrahepatic bile ducts (pIHBDs) to determine whether hepatocytes
can transdifferentiate into normal cholangiocytes and
assemble into functional bile ducts. In our ALGS mouse model
genes encoding the NOTCH DNA binding partner RBP and the
biliary transcription factor HNF6 are inactivated in embryonic
liver progenitors by Cre-mediated recombination. Like humans
with severe ALGS these mice lack pIHBDs at birth; remarkably,
however, pIHBDs are formed in our ALGS mouse model with
age. To determine whether hepatocytes are the source of the
new pIHBDs in our Cre-based mouse model, we developed a
Flp recombinase-mediated method of hepatocyte fate tracing.
By following fate-traced hepatocytes through de novo pIHBD
formation we determined that hepatocytes convert into cholangiocytes
and undergo tubulogenesis. This results in a fully functional
hepatocyte-derived biliary system capable of reverting
cholestasis and liver injury. We found little clonal expansion of
hepatocyte-derived cholangiocytes, which suggests that these
cells do not transition through a transit amplifying cell stage,
but rather many hepatocytes transdifferentiate and are incorporated
into the new pIHBDs. Interestingly, we observed that
biliary markers are activated in the hepatocytes near hypoxic
regions, suggesting that local hypoxia may induce hepatocyte
bile duct formation. Indeed, we show that activation of hypoxia
signaling in hepatocytes in vivo can induce biliary differentiation.
Our results demonstrate that hepatocytes can generate
an intrahepatic biliary system effective in relieving liver injury
in a mouse model of severe ALGS. These results define the
plasticity of hepatocytes and add another dimension to the
remodeling capacity of the adult liver. In addition, hepatocyte
transdifferentiation independent of NOTCH signaling in our
ALGS model suggests the existence of alternative drivers of
biliary differentiation, like hypoxia signaling. Bile duct formation
by hepatocytes may explain spontaneous improvement of
cholestasis observed in some ALGS patients and could potentially
be developed into an in vivo-reprogramming-based therapy
for others.
Disclosures:
Johanna R. Schaub - Management Position: MDsave
The following authors have nothing to disclose: Kari A. Huppert, Ashley E. Cast,
Feng Chen, Stacey S. Huppert, Holger Willenbring
681
Identification of hepatic stem cell niche by label retaining
cell assay with fetal and neonatal mice
Reiichiro Kuwahara 1 , Neil D. Theise 2 , Takuji Torimura 1 ; 1 Department
of Medicine, Division of Gastroenterology, Kurume University
School of Medicine, Kurume, Japan; 2 Department of Pathology
and Medicine, Beth Israel Medical Center of Albert Einstein College
of Medicine, New York, NY
Background and Aim: Previously we showed, in an acetaminophen
injury model, that oval cells (OVc) appear to derive from
the most proximal portions of the biliary tree (canals of Hering:
CoH) in a time- and dose-dependent manner, and regenerating,
differentiated peribiliary hepatocytes (PbH) also contributed
to parenchymal repair (Kofman et al, Hepatology 2005;
41: 1252). Subsequent label retaining cell (LRC) assays supported
these findings (Kuwahara et al, Hepatology 2008; 47:
1994). We have now investigated whether LRC assays could
shed light on the possible role of CoH and PbH in normal liver
development, in the absence of injury. Methods: Pregnant mice
received water containing BrdU for scheduled term of 3 days
(E10-12, E13-15, and E16-19). For the confirmation of BrdU
labeling in the fetal liver, some mice were sacrificed immediately
after each labeling term. The other mice were allowed to
give birth to mice. Neonatal mice were raised to 8 weeks old
to “chase” for label washout and then sacrificed. Other mice
received intraperitoneal injection of BrdU for labeling at the
age of 6, 13, 20, 27, 34, 41, 48 and 55 days. Then, some
of them were sacrificed at 24 hours after each injection for the
analyses of cell division. Other mice were raised to 8 weeks
old to “chase” for label washout following the BrdU labeling
and then sacrificed. PbH and biliary cell in CoH were analyzed
by double immunostaining for biliary keratins (PanK)/
BrdU (cell division), PanK/Ki-67 (proliferation). Results: Almost
all cells in the fetal liver were labelled with maternally administered
BrdU in drinking water. In all mice with BrdU labeling
during pregnancy, neither label retaining PbH nor label retaining
biliary cells in CoH were detected in the liver of 8 weeks.
The Ki67 index of neonatal mice liver showed that frequencies
of cell proliferation of hepatocytes and biliary cells in CoH
were highest at the age of 7 days and then diminished steadily.
At 8 weeks post “chase”, distributions of BrdU-retaining PbH
and biliary cells in CoH to total BrdU positive cells (lobular and
peribiliary hepatocytes + biliary cells in CoH) were highest in
the liver with BrdU labeling at the age of 13 days. These distributions
were significant higher than those of liver with labeling
at the age of 6, 20 and 27 days, respectively. Conclusion:
These data provides support that PbH and CoH cells serve resident
stem cell functions including contributing to parenchymal
mass during organogenesis and early post-natal development
(< 3weeks), but not after.
Disclosures:
The following authors have nothing to disclose: Reiichiro Kuwahara, Neil D.
Theise, Takuji Torimura
682
Disruption of TGF-β-regulated CTCF Suppression of
Telomerase links a Human Stem Cell Disorder to Liver
Tumorigenesis
Jian Chen 1 , Jiun-Sheng Chen 1 , Young Jin Gi 1 , H. Franklin Herlong 1 ,
Yun Seong Jeong 1 , Nipun Mistry 1 , Xiaoping Su 1 , Asif Rashid 1 ,
Bibhuti Mishra 1 , Jon White 2 , Milind Javle 1 , Marta L. Davila 1 , John
R. Stroehlein 1 , Rosanna Weksbergc 3 , Jerry W. Shay, 4 , Keigo
Machida 5 , Hidekazu Tsukamoto 5 , Lopa Mishra 1 ; 1 The University
of Texas MD Anderson Cancer Center, Houston, TX; 2 Institute of
Clinical Research, Veterans Affairs Medical Center, Washington
DC, DC; 3 Hospital for Sick Children, Toronto, ON, Canada; 4 The
University of Texas Southwestern Medical Center, Dallas, TX; 5 University
of Southern California, Los Angeles, CA
Patients with a human stem cell disorder, the Beckwith-Wiedemann
syndrome (BWS) are known to develop multiple liver
tumor types (hepatoblastoma, hepatocellular cancer, and cholangiocarcinoma)
within a single patient. These patients are at
an 800 fold increased risk of tumorigenesis. However, a precise
mechanism for the switch in tumorigenesis remains elusive.
In a previous study we demonstrated that loss of Transforming
Growth Factor-beta (TGF-β) signaling, was a causal factor in
BWS. To determine the mechanism for the oncogenic switch,
we performed a broad bioinformatics and functional analyses
utilizing human BWS cells, tissues and our mutant models in the
TGF-β pathway. Methods and Results: (1) Over 80% of TGF-β/
Sptbn1 +/− /Smad3 +/− mutant mice spontaneously developed
multiple tumors including the liver tumors that were phenotypically
similar to those of patients with BWS. (2) Somatic mutations
in SPTBN1 and SMAD3 are among the most frequent in
human HCCs. (3) Both Sptbn1 +/− /Smad3 +/− mice and BWS
cells express increased levels of stem cell-associated genes,