studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 547A
including Nanog, Sox2 and Oct4. (4) Increased levels of several
stem-ness genes and telomerase (TERT) (25-45 fold), c-Myc
(20-100 fold), IGF2 (~20 fold), with decreased levels of molecules
implicated in BWS- P57 and CTCF were observed in liver
tumors that developed in Sptbn1 +/- /Smad3 +/- mice. (5) Disruption
of the β2SP/SMAD3/CTCF complex raises TERT levels
in BWS and in Sptbn1 +/− /Smad3 +/− mice. (6) We observed
that disruption of the β2SP/SMAD3/CTCF complex increases
stem-like properties (increased ALDH-positive population and
Sphere formation) and enhances tumorigenesis in human HCC
cell lines. (7) We identified a mechanism by which the long
range chromatin modulator CTCF facilitates TGF-β-mediated
repression of hTERT transcription via β2S/SMAD3/CTCF interactions.
Conclusions: We present new insights into liver tumor
formation through the human stem cell disorder BWS and our
mutant models in the TGF-β pathway, demonstrating a potential
shift arising from disruption of TGF-β/CTCF signaling leading
to BWS-associated tumorigenesis, telomerase activation,
and human stem cell associated cancer development. This syndrome
can provide new strategies for preventing and targeting
liver cancers.
Disclosures:
The following authors have nothing to disclose: Jian Chen, Jiun-Sheng Chen,
Young Jin Gi, H. Franklin Herlong, Yun Seong Jeong, Nipun Mistry, Xiaoping
Su, Asif Rashid, Bibhuti Mishra, Jon White, Milind Javle, Marta L. Davila, John
R. Stroehlein, Rosanna Weksbergc, Jerry W. Shay,, Keigo Machida, Hidekazu
Tsukamoto, Lopa Mishra
683
Self-renewing diploid Axin2+ cells fuel homeostatic
renewal of the liver
Bruce M. Wang 1 , Roel Nusse 2,3 ; 1 Medicine, UCSF Liver Center,
San Francisco, CA; 2 Developmental Biology, Stanford University,
Stanford, CA; 3 Howard Hughes Medical Institute, Stanford, CA
The cellular source of new hepatocytes in the adult liver and
the molecular regulation of hepatocyte renewal are fundamental
unanswered questions in liver biology. While it has been
shown that new hepatocytes in the uninjured liver arise from
pre-existing hepatocytes, hepatocytes are known to be heterogenous
with striking differences in age and function across
the liver lobule. It is unknown whether a specific subpopulation
of hepatocytes serves homeostatic renewal in the liver. Using
a novel mouse model for stably labeling Wnt-responsive cells
in vivo, we have discovered a unique population of Wnt-controlled
hepatocytes with stem cell characteristics surrounding
the central vein. These pericentral cells express the early liver
progenitor marker Tbx3, proliferate at a faster rate than other
hepatocytes and are diploid, and thus differ from mature
hepatocytes, which are mostly polyploid. Over time, these
cells self-renew and give rise to descendants that differentiate
into Tbx3-negative, polyploid hepatocytes and can replace all
hepatocytes along the liver lobule during homeostatic renewal.
Importantly, these Wnt-responsive cells are present in the normal
liver, thereby distinguishing them from injury-induced multipotent
progenitor cells (oval cells) associated with the portal
region. Adjacent central vein endothelial cells provide essential
Wnt signals that maintain the pericentral cell population,
thereby constituting the hepatocyte stem cell niche. Thus, we
describe for the first time a subset of hepatocytes that subserves
homeostatic hepatocyte renewal, characterize its anatomical
niche, and identify molecular signals that regulate its activity.
Disclosures:
The following authors have nothing to disclose: Bruce M. Wang, Roel Nusse
684
FAP disease modelling using patient-specific iPS cells
derived from urine
Christoph J. Niemietz, Vanessa Sauer, Jacquelline Stella, Gursimran
Chandhok, Andree Zibert, Hartmut H. Schmidt; Klinik für
Transplantationsmedizin, Universitätsklinikum Münster, Münster,
Germany
Transthyretin-related familial amyloid polyneuropathy (TTR-
FAP) is a rare genetic neurodegenerative disease caused by
mutations of TTR. TTR is primarily secreted by the liver and
misfolding ultimately leads to aggregation and fibril deposition
in peripheral tissues and organs. In order to prevent expression
of TTR, gene silencing strategies are currently under clinical
investigation. The use of primary cells derived from FAP
patients for evaluation of antisense strategies is difficult. As
an alternate, stem cell technology is used here to generate
patient specific primary cells. Urine collections of FAP patients
were processed for isolation of renal epithelial cells, followed
by reprogramming into iPS cells (iPSCs) using non-integrating
episomal vectors. After characterization of pluripotent cell lines,
differentiation towards hepatocyte-like cells was accomplished
after treatment with factors for 14 days. iPSC-Heps were characterized
by analysis of typical hepatic markers via qRT-PCR,
flow cytometry, and immunocytochemistry. ASOs and siRNAs
were introduced into iPSC-Heps in order to evaluate gene
silencing of TTR via qRT-PCR analysis, ELISA and Western-blot.
Typically, 2-3 stable cell colonies emerged upon cultivation
of urine-derived cells from FAP patients (n=12). iPSC-Heps
showed similar morphology and gene expression in comparison
to HepG2 control. TTR gene expression was high. ASOs or
siRNA treatment resulted in downregulation of TTR. Our results
indicate that iPSCs derived from urine cells of FAP patients can
be routinely reprogrammed and differentiated into iPSC-Heps
expressing TTR at comparable levels as control cells. TTR gene
silencing analysis of iPSC-Heps allows the establishment of a
patient-specific in vitro platform for evaluating drug efficiency.